Compositions and methods for detecting small rnas, and uses thereof

ABSTRACT

Compositions and methods are provided for the detection of small RNA target nucleic acids, preferably miRNA target nucleic acids, wherein the compositions and methods provide for sensitive and specific detection of the target nucleic acids. The compositions and methods include using one or more of a first amplification oligomer that is preferably an extender primer, a target capture oligomer that is preferably at least partially double stranded, a promoter primer/provider, a reverse primer that is preferably a universal primer and a detection probe. The compositions and methods are useful for diagnostics, prognostics, monitoring the effectiveness of treatment and/or determining a treatment.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. 119(e) ofprovisional application No. 61/117,913, filed Nov. 25, 2008, which isincorporated by reference herein.

FIELD OF THE INVENTION

The invention is related to the field of nucleic acid assays, andparticularly to detection of specific target small nucleic acid presentin a sample, such a small RNA or DNA sequence present in a biologicalsample.

BACKGROUND

Detection of nucleic acids in a sample is useful in diagnostic,therapeutic, forensic, agricultural, food science applications and otherareas. Methods of nucleic acid detection generally include those thatuse physical separation of a nucleic acid, such as by capturing thenucleic acid in or on a matrix or support followed by detecting thepresence of captured nucleic acids and/or amplification products madetherefrom. Detection can take place by a number of methods known in theart, such as mass spectrometry, sequencing, a dye or intercalatingagent, or by hybridizing a detectable probe to the nucleic acid. Somemethods indirectly detect nucleic acids by producing a product made fromusing a target nucleic acid as a template and detecting the product,e.g., detecting an RNA transcript made from a DNA, or a translatedprotein made from an RNA transcript. Other indirect methods detect aproduct made by an enzymatic reaction associated with the nucleic acidto be detected, e.g., an enzyme-linked probe hybridized to the targetnucleic acid which produces a detectable response when the enzyme'ssubstrate is provided. Some methods of nucleic acid detection rely onamplifying a nucleic acid sequence to produce a larger quantity ofnucleic acid that is detected. Examples of amplification methods includeproducing many copies of a cloned sequence and in vitro amplificationprocedures that use enzymatic synthesis of multiple copies of a nucleicacid sequence.

Many of the techniques for detecting nucleic acids require the presenceof a relatively large amount or proportion of the target nucleic acid inthe sample, while other techniques use nucleic acid amplification toincrease the amount or proportion of the nucleic acid to be detectedfrom a smaller amount of the target nucleic acid in a sample. Enrichmentof some or all of the nucleic acid present in a sample may facilitatedetection of the nucleic acid of interest. Many known procedures fornucleic acid enrichment and detection are laborious, time-consuming, orrequire use of equipment or hazardous chemicals (e.g., chaotropes,mutagens, or radioactive compounds) that make such proceduresundesirable for many applications, such as for rapid screening of manysamples, point-of-care diagnostics, or detection at a site outside of alaboratory. Thus, there remains a need for a method that providesrelatively simple procedures and sufficient sensitivity and/orspecificity to detect a nucleic acid of interest.

The physical nature or relative abundance of some nucleic acids mayimpede their detection in a sample. For example, small RNA (about 17-27nt), such as microRNA (miRNA), small or short interfering RNA (siRNA),short hairpin RNA (shRNA), and small nuclear RNA (snRNA) are difficultto separate from other sample components and/or to detect by using knownmethods. Small RNA are often relatively rare in a biological sample,which contributes to the difficulty of their detection. Furthercomplicating the detection of these small RNA molecules is that theseshort template RNA species often share high sequence homology betweenclosely related family members. Most small RNA detection methods in theart use general RNA isolation techniques, like Invitrogen Trizolreagent, Stratagene Total RNA, ABI mirVana and ABI Total RNA,(Invitrogen, Carlsbad, Calif.; Stratagene, La Jolla, Calif.; ABI, FosterCity Calif.), all of which provide an indiscriminant abundance of RNAspecies that may include the target small RNA molecule(s) and includemany other non-target nucleic acids. These non-target nucleic acidsoften interfere with analysis of target sequence. These one size fitsall approaches introduce bias and experimental error into small RNAexpression profiling experiments. Because small RNA are regulatorymolecules that modulate or silence gene expression via RNA interference(RNAi), they are important molecules for understanding biologicalprocesses and disease states and as disease preventive or therapeuticagents. Studies have shown that differential small RNA expression occursin cancerous and non-cancerous cells. Thus, there is a need forcompositions and methods that rapidly and accurately isolate small RNAfrom a sample for downstream analysis. There is a need to rapidly andaccurately isolate and detect the presence of one or more small RNA inbiological samples to determine their abundance, relative abundance,expression level, stability, therapeutic efficacy, or othercharacteristics in a biological sample. There is a further need torapidly and accurately detect relative amounts of differentiallyexpressed small RNA in a variety of biological samples as an indicatorof a cancerous condition and/or as a part of a cancer prognosis bydetermining metastatic potential of a tumor and in developing a suitabletreatment thereof.

Current methods for detecting small RNA in biological samples are timeconsuming and imprecise. Common techniques include PCR (RT and qRT), insitu hybridization, nuclease protection assays, Northern blots to detectRNA, Western blots, immunoassays, and fluorescence detection assays (PCTApp. Nos. WO 00/44914, Li et al., WO 05/04794, Bumcrot et al.).Detection methods also exist wherein an additional sequences is added tothe small RNA to facilitate priming and detection, such as adding asingle universal extension primer or adapter to every miRNA in a sample(Chen et al. U.S. Pat. No. 7,601,495; Raymond et al. RNA, 11:1737-44(2005)). These added nucleic acid sequences are then used as primerbinding sites for amplification of the small RNA and/or as captureprobe-binding sites, (Mullinax et al., US Pat. Pub. No. 2008-0182235;Jacobsen et al. WO 05/098029). In addition, these added nucleic acidsequences can be used as primers for a first amplification (Chen et al.Nuc. Acid. Res., 33(20):e179 (2005)). Another method for amplifying anddetecting a small RNA includes using a bridging oligo that iscomplementary to both a small RNA molecules and a unique nucleic acidmolecule followed by a ligating step to join the small RNA and theunique nucleic acid (Yeakley, US Pat. Pub. No. 2006-0019258). Thesemethods, unfortunately, are inaccurate; particularly as throughputincreases so too does the variability in result data (e.g., Mestdagh,Chen et al. Nuc. Acid. Res. 36(21):e143 (2008) and Nelson et al.,Biochim. Biophys Acta, 1179(11):758-65 (2008)). Thus, the methods in theart are not fully sufficient for providing reliable small RNA expressiondata for use in diagnosing disorders in which one or more of these smallRNAs play a role. Moreover, sample-processing methods in the art arecrude and inadequate for sample-to-answer automation, for identifyinghigh throughput processing of a large number of specimens, particularlyfor biomarker validation and diagnostic applications. There is a needfor a method for accurately detecting the presence and relativeabundance of one or more small RNAs in a sample for diagnosing andmonitoring a disorder, as well as for prognosing the disorder and/ormonitoring the efficacy of a treatment of the disorder. There is also aneed for methods and reagents useful with high throughput analysis ofand diagnostics using biomarkers, such as small RNA.

This application responds to the need for rapid, accurate and efficientnucleic acid detection assays by disclosing methods and compositionsuseful for the isolation and detection of one or more nucleic acids insamples, including one or more small RNA in biological samples, and thatare amenable with fully automated platforms.

SUMMARY OF THE INVENTION

An aspect of the invention is a method of detecting the presence of anucleic acid present in a sample which includes the steps of: providinga sample containing a small RNA target nucleic acid that is a micro RNA,miR-21, miR-34b, miR-182, miR-221, miR-222 or miR-802, mixing the samplewith a nucleic acid capture probe that forms under hybridizingconditions a partially double-stranded hairpin structure made up of aninternal target hybridizing sequence, flanked by a capture region, and aterminal region that binds to the capture region to form adouble-stranded stem portion of the hairpin structure in whichthe,target hybridizing region forms a substantially single-stranded loopportion, specifically hybridizing the target hybridizing sequence of thecapture probe to a target sequence in the target nucleic acid, bindingthe capture region to an immobilized probe attached to a capture supportby binding together members of a specific binding pair, thereby forminga capture hybrid made up of the target nucleic acid, the capture probe,and the immobilized probe attached to the capture support, separatingthe capture hybrid attached to the capture support from samplecomponents, releasing the target nucleic acid from the capture hybrid,then specifically hybridizing a detection probe to the target nucleic toform a detection hybrid, and detecting a signal produced from thedetection hybrid to indicate the presence of the target nucleic acid inthe sample. In one embodiment, the capture region is located near the 3′end of the capture probe and the terminal region is located near the 5′end of the capture probe. In another embodiment, the capture region islocated near the 5′ end of the capture probe and the terminal region islocated near the 3′ end of the capture probe. In one embodiment, thestep of binding the capture region to the immobilized probe hybridizescomplementary sequences of the capture region and the immobilized probe.In another embodiment, binding the capture region to the immobilizedprobe binds together non-nucleic acid members of a specific bindingpair, such as a ligand and its receptor. In one embodiment, releasingthe target nucleic acid from the capture hybrid further releases thecapture probe from the immobilized probe. In one embodiment, anamplification step uses a first amplification oligomer that specificallyhybridizes to all or a portion of the same target sequence that thetarget capture probe hybridizes. In one embodiment, an amplificationstep uses a promoter based amplification oligomer that specificallyhybridizes to a target sequence that is complementary to all or aportion of the target sequence that the target capture probe hybridizes.In one embodiment, the detecting step uses a detection probe thathybridizes specifically to a target sequence that is the same targetsequence that hybridizes to the target hybridizing sequence of thecapture probe. In another embodiment, the detection probe hybridizesspecifically to a target sequence that differs from or overlaps thetarget sequence that hybridizes to the target hybridizing sequence ofthe capture probe. In a preferred embodiment, the detecting step detectsa signal is produced in a homogeneous reaction.

Another aspect of the invention is a method of detecting the presence ofa target nucleic acid present in a sample that includes the steps of:providing a sample containing a small RNA target nucleic acid that is amicro RNA, miR-21, miR-34b, miR-182, miR-221, miR-222 or miR-802, mixingthe sample with a capture probe that is at least a partiallydouble-stranded structure made up of a first strand and a second strandof nucleic acid, wherein the first strand includes a target hybridizingregion and a capture region, and the second strand contains a sequencecomplementary to a sequence of the first strand, specificallyhybridizing the target hybridizing region of the capture probe to atarget sequence in the target nucleic acid, binding the capture regionto an immobilized probe attached to a capture support, thereby forming acapture hybrid made up of the target nucleic acid, the first strand ofthe capture probe, and the immobilized probe attached to the capturesupport, separating the capture hybrid attached to the capture supportfrom other sample components, releasing the target nucleic acid from thecapture hybrid, then specifically hybridizing a detection probe to thetarget nucleic acid to form a detection hybrid, and detecting a signalproduced from the detection hybrid, thereby indicating the presence ofthe target nucleic acid in the sample. In one embodiment, the firststrand Contains a 5′ capture region covalently linked to a 3′ targethybridizing region, and the second strand contains a 3′ sequencecomplementary to the capture region of the first strand, thereby forminga partially double-stranded structure when the capture region of thefirst strand hybridizes to the complementary 3′ sequence of the secondstrand. In another embodiment, the first strand contains a 5′ targethybridizing region covalently linked to a 3′ capture region, and thesecond strand contains a 5′ sequence complementary to the 3′ captureregion of the first strand, thereby forming a partially double-strandedstructure when the capture region of the first strand hybridizes to thecomplementary 5′ sequence of the second strand. In one embodiment, anamplification step uses a first amplification oligomer that specificallyhybridizes to all or a portion of the same target sequence that thetarget capture probe hybridizes. In one embodiment, an amplificationstep uses a promoter based amplification oligomer that specificallyhybridizes to a target sequence that is complementary to all or aportion of the target sequence that the target capture probe hybridizes.

Another aspect of the invention is a nucleic acid capture probe thatforms at least a partially double-stranded structure under hybridizingconditions and includes a target hybridizing sequence and a captureregion that binds to an immobilized probe by using members of a specificbinding pair. In one embodiment, the partially double-stranded structureis a hairpin structure made up of a contiguous linear sequence thatincludes an internal target hybridizing sequence, flanked by the captureregion and a terminal region that binds to the capture region to form adouble-stranded stem portion of the hairpin structure and the targethybridizing region forms a substantially single-stranded loop portion ofthe hairpin structure. It will be appreciated that the capture regionmay be a 5′ region and the terminal region is a 3′ region of thecontiguous linear sequence, or alternatively, the capture region may bea 5′ region and the terminal region is a 3′ region of the contiguouslinear sequence that forms a hairpin structure. In another capture probeembodiment, the structure is made of up of a first strand that includesthe target hybridizing region and the capture region, and a separatesecond strand that includes a sequence complementary to a sequence ofthe first strand such that hybridization of the complementary sequencesof the first strand and second strand produce at least a partiallydouble-stranded structure. It will be appreciated that the first strandmay have a 5′ target hybridizing region and a 3′ capture region, oralternatively, a 3′ target hybridizing region and a 5′ capture region,and that the complementary sequence of the separate second strand may becomplementary to either the target hybridizing region or the captureregion of the first strand. In a preferred embodiment, the complementarysequence of the second strand is complementary to the capture region ofthe first strand.

One embodiment is a method of detecting the presence of a target nucleicacid in a sample comprising the steps of: providing a target captureprobe that forms under hybridizing conditions a partiallydouble-stranded hairpin structure made up of an internal targethybridizing sequence flanked by a capture region and a terminal region,wherein the terminal region binds to the capture region to form adouble-stranded stem portion of the hairpin structure, and wherein thetarget hybridizing region forms a single-stranded loop portion of thehairpin structure; mixing a sample suspected of containing a small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidwith the capture probe; specifically hybridizing the target hybridizingsequence of the capture probe to a target sequence in the small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleicacid; binding the capture region to an immobilized probe attached to acapture support by binding together members of a specific binding pair,thereby forming a capture hybrid made up of the small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid, thecapture probe, and the immobilized probe attached to the capturesupport; separating the capture hybrid attached to the capture supportfrom other components in the sample; releasing the small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid fromthe capture hybrid; providing to the mixture comprising the releasedsmall RNA, miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 targetnucleic acid and the target capture probe at least a first amplificationoligomer comprising a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe target capture probe; providing a set of conditions wherein thefirst amplification oligomer hybridizes to the small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid and anamplification reaction takes place, and wherein the target capture probeis in a partially double stranded hairpin formation; generating adetectable amplification product in an amplification reaction whereinthe first amplification oligomer generates a cDNA strand from the smallRNA, miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleicacid; and detecting the amplification product to indicate presence ofsmall RNA, miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 targetnucleic acid in the sample.

Another embodiment is a method of detecting the presence of a targetnucleic acid in a sample comprising the steps of: providing a targetcapture probe that forms under hybridizing conditions a partiallydouble-stranded hairpin structure made up of an internal targethybridizing sequence flanked by a capture region and a terminal region,wherein the terminal region binds to the capture region to form adouble-stranded stem portion of the hairpin structure, and wherein thetarget hybridizing region forms a single-stranded loop portion of thehairpin structure; mixing a sample suspected of containing a small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidwith the capture probe; specifically hybridizing the target hybridizingsequence of the capture probe to a target sequence in the miR-221 targetnucleic acid; binding the capture region to an immobilized probeattached to a capture support by binding together members of a specificbinding pair, thereby forming a capture hybrid made up of the small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleicacid, the capture probe, and the immobilized probe attached to thecapture support; separating the capture hybrid attached to the capturesupport from other components in the sample; releasing the small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidfrom the capture hybrid and providing a set of conditions wherein thetarget capture probe is a partially double-stranded hairpin structure;providing to the mixture comprising the released small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid andthe target capture probe a plurality of amplification oligomers, theplurality comprising at least a first amplification oligomer and apromoter-based oligomer, wherein the first amplification oligomercomprises a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe target capture probe, and wherein the promoter-based oligomercomprises a target hybridizing sequence, a portion of which issubstantially complementary to a portion of the target hybridizingregion of the target capture probe; providing a set of conditionswherein the first amplification oligomer hybridizes to the small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidin the presence of the hairpin capture probe; performing anamplification reaction using the plurality of amplification oligomers togenerate amplification product from the small RNA, miR21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acid; and detectingthe amplification product to indicate presence of small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid in thesample.

Another embodiment is a multiplex amplification method for specificallydetecting the presence of target nucleic acids in a sample comprisingthe steps of: providing a first target capture probe for capturing asmall RNA, miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 targetnucleic acid and a second target capture probe for capturing a smallRNA, miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleicacid, wherein the target capture probes are configured to targetdifferent target nucleic acids, wherein the target capture probes areeach made up of an internal target hybridizing sequence flanked by acapture region and a terminal region, wherein the terminal region bindsto the capture region to form a double-stranded stem portion of thehairpin structure, wherein the target hybridizing sequence forms asingle-stranded loop portion of the hairpin structure, and wherein thetarget hybridizing sequence of the capture probe is substantiallycomplementary to all or a portion of a small RNA, miR21, miR-34b,miR-182, miR-221, miR-222 or miR-802 sequence and target hybridizingsequence of the miR-182 target capture probe is substantiallycomplementary to all or a portion of a small RNA, miR21, miR-34b,miR-182, miR-221, miR-222 or miR-802; mixing a sample suspected ofcontaining two or more of a small RNA, miR21, miR-34b, miR-182, miR-221,miR-222 or miR-802 target nucleic acid with the capture probes;specifically hybridizing the target hybridizing sequence of the captureprobes to a target sequence in their respective target nucleic acids;binding the capture regions to an immobilized probe attached to acapture support by binding together members of a specific binding pair,thereby forming capture hybrids made up of the small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid, thefirst capture probe and the immobilized probe attached to the capturesupport, and another capture hybrid made up of small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acid, thesecond capture probe, and the immobilized probe attached to the capturesupport; separating the capture hybrids from other components in thesample; releasing the target nucleic acids from their capture hybridsand providing a set of conditions wherein the first and second targetcapture probes are partially double-stranded hairpin structures;providing to the mixture comprising the released target nucleic acidsand the target capture probes a plurality of amplification oligomers,the plurality comprising, (i) at least a first amplification oligomerand a promoter-based oligomer for specifically hybridizing to small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidcaptured by the first target capture probe or cDNA strand thereof,wherein the first amplification oligomer comprises a target hybridizingsequence, a portion of which is substantially identical to a portion ofthe target hybridizing region of the first target capture probe, andwherein the promoter-based oligomer comprises a target hybridizingsequence, a portion of which is substantially complementary to a portionof the target hybridizing region of the first target capture probe, and(ii) at least a first amplification oligomer and a promoter-basedoligomer for hybridizing to a small RNA, miR21, miR-34b, miR-182,miR-221, miR-222 or miR-802 target nucleic acid captured by the secondtarget capture probe or cDNA strand thereof, wherein the firstamplification oligomer comprises a target hybridizing sequence, aportion of which is substantially identical to a portion of the targethybridizing region of the second target capture probe, and wherein thepromoter-based oligomer comprises a target hybridizing sequence, aportion of which is substantially complementary to a portion of thetarget hybridizing region of the second target capture probe; providinga set of conditions wherein the first amplification oligomers hybridizeto their respective target nucleic acids; performing an amplificationreaction using the plurality of amplification oligomers to generateamplification products from the small RNA, miR21, miR-34b, miR-182,miR-221, miR-222 or miR-802 and miR-182 target nucleic acids captured bythe first and second target capture probes; and detecting theamplification products to indicate presence of a small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidcaptured by the first target capture probe, and small RNA, miR21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidcaptured by the first target capture probe or both in the sample. Themultiplex methods can similarly include more than two target nucleicacids, and the oligomers used will increase accordingly.

Another embodiment is a method of detecting the presence of a targetnucleic acid in a sample comprising the steps of: providing a targetcapture probe that forms under hybridizing conditions a partiallydouble-stranded hairpin structure made up of an internal targethybridizing sequence flanked by a capture region and a terminal region,wherein the terminal region binds to the capture region to form adouble-stranded stem portion of the hairpin structure, and wherein thetarget hybridizing region forms a single-stranded loop portion of thehairpin structure; mixing a sample suspected of containing a miR-221target nucleic acid with the capture probe; specifically hybridizing thetarget hybridizing sequence of the capture probe to a target sequence inthe miR-221 target nucleic acid; binding the capture region to animmobilized probe attached to a capture support by binding togethermembers of a specific binding pair, thereby forming a capture hybridmade up of the miR-221 target nucleic acid, the capture probe, and theimmobilized probe attached to the capture support; separating thecapture hybrid attached to the capture support from other components inthe sample; releasing the miR-221 target nucleic acid from the capturehybrid; providing to the mixture comprising the released miR-221 targetnucleic acid and the target capture probe at least a first amplificationoligomer comprising a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe target capture probe; providing a set of conditions wherein thefirst amplification oligomer hybridizes to the miR-221 target nucleicacid and an amplification reaction takes place, and wherein the targetcapture probe is in a partially double stranded hairpin formation;generating a detectable amplification product in an amplificationreaction wherein the first amplification oligomer generates a cDNAstrand from the miR-221 target nucleic acid; and detecting theamplification product to indicate presence of miR-221 target nucleicacid in the sample.

Another embodiment is a method of detecting the presence of a targetnucleic acid in a sample comprising the steps of: providing a targetcapture probe that forms under hybridizing conditions a partiallydouble-stranded hairpin structure made up of an internal targethybridizing sequence flanked by a capture region and a terminal region,wherein the terminal region binds to the capture region to form adouble-stranded stem portion of the hairpin structure, and wherein thetarget hybridizing region forms a single-stranded loop portion of thehairpin structure; mixing a sample suspected of containing a miR-221target nucleic acid with the capture probe; specifically hybridizing thetarget hybridizing sequence of the capture probe to a target sequence inthe miR-221 target nucleic acid; binding the capture region to animmobilized probe attached to a capture support by binding togethermembers of a specific binding pair, thereby forming a capture hybridmade up of the miR-221 target nucleic acid, the capture probe, and theimmobilized probe attached to the capture support; separating thecapture hybrid attached to the capture support from other components inthe sample; releasing the miR-221 target nucleic acid from the capturehybrid and providing a set of conditions wherein the target captureprobe is a partially double-stranded hairpin structure; providing to themixture comprising the released miR-221 target nucleic acid and thetarget capture probe a plurality of amplification oligomers, theplurality comprising at least a first amplification oligomer and apromoter-based oligomer, wherein the first amplification oligomercomprises a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe target capture probe, and wherein the promoter-based oligomercomprises a target hybridizing sequence, a portion of which issubstantially complementary to a portion of the target hybridizingregion of the target capture probe; providing a set of conditionswherein the first amplification oligomer hybridizes to the miR-221target nucleic acid in the presence of the hairpin capture probe;performing an amplification reaction using the plurality ofamplification oligomers to generate amplification product from themiR-221 target nucleic acid; and detecting the amplification product toindicate presence of miR-221 target nucleic acid in the sample.

Another embodiment is a multiplex amplification method for specificallydetecting the presence of target nucleic acids in a sample comprisingthe steps of: providing a target capture probe for capturing a miR-221target nucleic acid and a target capture probe for capturing a miR-182target nucleic acid, wherein the target capture probes are each made upof an internal target hybridizing sequence flanked by a capture regionand a terminal region, wherein the terminal region binds to the captureregion to form a double-stranded stem portion of the hairpin structure,wherein the target hybridizing region forms a single-stranded loopportion of the hairpin structure, and wherein the target capture regionof the miR-221 target capture probe is substantially complementary toall or a portion of a miR-221 sequence and the target capture region ofthe miR-182 target capture probe is substantially complementary to allor a portion of a miR-182 sequence; mixing a sample suspected ofcontaining a miR-221 target nucleic acid and a miR-182 target nucleicacid with the capture probes; specifically hybridizing the targethybridizing sequence of the capture probes to a target sequence in theirrespective target nucleic acids; binding the capture regions to animmobilized probe attached to a capture support by binding togethermembers of a specific binding pair, thereby forming a capture hybridmade up of the miR-221 target nucleic acid, the capture probe and theimmobilized probe attached to the capture support, and a capture hybridmade up of the miR-182 target nucleic acid, the capture probe, and theimmobilized probe attached to the capture support; separating thecapture hybrids from other components in the sample; releasing themiR-221 and miR-182 target nucleic acids from the capture hybrids andproviding a set of conditions wherein the target capture probes arepartially double-stranded hairpin structures; providing to the mixturecomprising the released miR-221 and miR-182 target nucleic acids and thetarget capture probes a plurality of amplification oligomers, theplurality comprising, (i) at least a first amplification oligomer and apromoter-based oligomer for specifically hybridizing to a miR-221 targetnucleic acid or cDNA strand thereof, wherein the first amplificationoligomer comprises a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe miR-221 target capture probe, and wherein the promoter-basedoligomer comprises a target hybridizing sequence, a portion of which issubstantially complementary to a portion of the target hybridizingregion of the miR-221 target capture probe, and (ii) at least a firstamplification oligomer and a promoter-based oligomer for hybridizing toa miR-182 target nucleic acid or cDNA strand thereof, wherein the firstamplification oligomer comprises a target hybridizing sequence, aportion of which is substantially identical to a portion of the targethybridizing region of the miR-182 target capture probe, and wherein thepromoter-based oligomer comprises a target hybridizing sequence, aportion of which is substantially complementary to a portion of thetarget hybridizing region of the miR-182 target capture probe; providinga set of conditions wherein the first amplification oligomers hybridizesto their respective target nucleic acids; performing an amplificationreaction using the plurality of amplification oligomers to generateamplification products from the miR-221 and miR-182 target nucleicacids; and detecting the amplification products to indicate presence ofmiR-221 target nucleic acid, miR-182 target nucleic acid or both in thesample.

In some aspects of the methods the capture region is joined to the 3′end of the target capture probe target hybridizing sequence. In someaspects, the capture region is joined to the 5′ end of the targetcapture probe target hybridizing sequence. In some aspects, binding thecapture region to the immobilized probe results from hybridizingcomplementary sequences of the capture region and the immobilized probe.In some aspects, the capture region is a nucleotide sequence selectedfrom the group consisting of: dT.sub.0-3A.sub.10-30, which means thatthe polyT portion of the capture region is from 0 to 3 nucleotides inlength and the polyA portion of the capture region is from 10 to 30nucleotide in length. In some aspects, the capture region is dA.sub.12.In some aspects, the step of releasing the target nucleic acid from thecapture hybrid further releases the capture probe from the immobilizedprobe.

In some aspects of the methods, the target capture probe specificallyhybridizes to a small RNA target nucleic acid. In some aspects, thetarget capture probe specifically hybridizes to a miR-21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acid. In someaspects, target capture probe specifically hybridizes to a targetnucleic acid and has a target hybridizing sequence that is at least 95%complementary to SEQ ID NO:44, 41, 47, 38, 29 or 34. In some aspects,the target capture probe specifically hybridizes to a miR-221 targetnucleic acid and has a target hybridizing sequence that is at least 95%identical to SEQ ID NO:24. In some aspects, target capture probespecifically hybridizes to a miR-221 target nucleic acid and has anucleotide sequence that is at least 95% complementary to SEQ ID NO:44.In some aspects, the target capture probe that specifically hybridizesto a miR-221 target nucleic acid is SEQ ID NO:24. In some aspects, thetarget capture probe that specifically hybridizes to a miR-21 targetnucleic acid is SEQ ID NO:22. In some aspects, the target capture probethat specifically hybridizes to a miR-182 target nucleic acid is SEQ IDNO:23. In some aspects, the target capture probe that specificallyhybridizes to a miR-222 target nucleic acid is SEQ ID NO:25. In someaspects, the target capture probe that specifically hybridizes to amiR-802 target nucleic acid is SEQ ID NO:26.

In some aspects of the methods the first amplification oligomer is anextender primer comprising a 3′ target hybridizing sequence and a 5′extension nucleic acid sequence, wherein the 3′ target hybridizingsequence is substantially complementary to about 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or more contiguousnucleic acids at the 3′ end of its respective target nucleic acid. Insome aspects of the methods the first amplification oligomer is anextender primer comprising a 3′ target hybridizing sequence and a 5′extension nucleic acid sequence, wherein the 3′ target hybridizingsequence is substantially complementary to about 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or more contiguousnucleic acids at the 3′ end of its respective small RNA target nucleicacid. In some aspects of the methods the first amplification oligomer isan extender primer comprising a 3′ target hybridizing sequence and a 5′extension nucleic acid sequence, wherein the 3′ target hybridizingsequence is substantially complementary to about 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or more contiguousnucleic acids at the 3′ end a target nucleic acid that is a miR-21,miR-34b, miR-182, miR-221, miR-222 or miR-802. In some aspects, thefirst amplification oligomer has an extension nucleic acid sequence thatis at least 95% identical to SEQ ID NO:49. In some aspects, the firstamplification oligomer targets miR-221 and has an extension nucleic acidsequence that is at least 95% identical to SEQ ID NO:49. In someaspects, the first amplification oligomer targets miR-182 and has anextension nucleic acid sequence that is at least 95% identical to SEQ IDNO:49. In some aspects, the first amplification oligomer is an extenderprimer targeting a miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802target nucleic acid and comprises a 3′ target hybridizing sequence and a5′ extension nucleic acid sequence, wherein the 3′ target hybridizingsequence is substantially identical to about 9 or more contiguousnucleic acids at the 5′ end of the target hybridizing sequence of atarget capture probe targeting miR2I, miR-34b, miR-182, miR-221, miR-222or miR-802. In some aspects, the first amplification oligomer is anextender primer targeting miR-221 and comprising a 3′ target hybridizingsequence and a 5′ extension nucleic acid sequence, wherein the 3′ targethybridizing sequence is substantially identical to about 9 or morecontiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-221 target capture probe. In some aspects, the firstamplification oligomer is an extender primer targeting miR-182 andcomprising a 3′ target hybridizing sequence and a 5′ extension nucleicacid sequence, wherein the 3′ target hybridizing sequence issubstantially identical to about 9 or more contiguous nucleic acids atthe 5′ end of the target hybridizing sequence of the miR-182 targetcapture probe. In some aspects, the extension nucleic acid sequence isat least 95% identical to SEQ ID NO:49. In some aspects, the targetcapture oligomer is SEQ ID NO:24 and the first amplification oligomer isSEQ ID NO:17.

In some aspects of the methods, a promoter based oligomer and a reverseprimer oligomer are provided to the mixture and participate in theamplification reaction. In some aspects, the promoter based oligomercomprises a target hybridizing sequence that is substantiallycomplementary all or a portion of the nucleic acids on 3′ end of thetarget capture probe target hybridizing region. In some aspects, thepromoter based oligomer comprises a target hybridizing sequence that issubstantially complementary to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or more contiguousnucleic acids on 3′ end of the target capture probe target hybridizingregion. In some aspects, the promoter based oligomer comprises a targethybridizing sequence that is substantially complementary to about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27 or more contiguous nucleic acids on 3′ end of the miR-21,miR-34b, miR-182, miR-221, miR-222 or miR-802 target capture probetarget hybridizing region. In some aspects, the promoter based oligomercomprises a target hybridizing sequence that is substantiallycomplementary to about 12 or more contiguous nucleic acids on 3′ end ofthe miR-221 target capture probe target hybridizing region. In someaspects, the promoter based oligomer comprises a target hybridizingsequence that is substantially complementary to about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 ormore contiguous nucleic acids on 3′ end of the target capture probetarget hybridizing region and the first amplification oligomer has anucleotide sequence that is at least 95% identical to SEQ ID NO:49. Insome aspects, the promoter based oligomer comprises a target hybridizingsequence that is substantially complementary to about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 ormore contiguous nucleic acids on 3′ end of the miR-21, miR-34b, miR-182,miR-221, miR-222 or miR-802 target capture probe target hybridizingregion and the first amplification oligomer has a nucleotide sequencethat is at least 95% identical to SEQ ID NO:49. In some aspects, thepromoter based oligomer comprises a target hybridizing sequence that issubstantially complementary to about 12 or more contiguous nucleic acidson 3′ end of the miR-221 target capture probe target hybridizing regionand the first amplification oligomer has a nucleotide sequence that isat least 95% identical to SEQ ID NO:49. In some aspects, the promoterbased oligomer comprises a target hybridizing sequence that issubstantially complementary all or a portion of the nucleic acids on 3′end of the target capture probe target hybridizing region and the targetcapture oligomer is SEQ ID NO:24. In some aspects, the promoter basedoligomer comprises a target hybridizing sequence that is substantiallycomplementary to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or more contiguous nucleic acidson 3′ end of the target capture probe target hybridizing region and thetarget capture oligomer is SEQ ID NO:24. In some aspects, the promoterbased oligomer comprises a target hybridizing sequence that issubstantially complementary to about 12 or more contiguous nucleic acidson 3′ end of the a target capture oligomer that is SEQ ID NO:24. In someaspects the promoter based amplification oligomer is SEQ ID NO:11. Insome aspects, the reverse primer targets a portion of a nucleic acidamplification product that is complementary to the extension nucleicacid sequence of the first amplification oligomer.

In some aspects of the method, the amplification reaction comprises afirst amplification oligomer configured to specifically hybridize atarget nucleic acid, and a reverse primer targets a complement of theextension nucleic acid sequence of the first amplification oligomer. Insome aspects, the amplification reaction comprises a first amplificationoligomer configured to specifically hybridize a target nucleic acid, anda universal reverse primer targets a complement of the extension nucleicacid sequence of the first amplification oligomer. In some aspects, theamplification reaction comprises a first amplification oligomerconfigured to specifically hybridize a target nucleic acid and whichcomprises an extension nucleic acid sequence that is substantiallyidentical to SEQ ID NO:49, and a reverse primer targets a complement ofthe extension nucleic acid sequence of the first amplification oligomer.In some aspects, the amplification reaction comprises a firstamplification oligomer configured to specifically hybridize a targetnucleic acid and which comprise an extension nucleic acid sequence thatis substantially identical to SEQ ID NO:49, and the reverse primer isSEQ ID NO:20. In some aspects, the amplification reaction comprises afirst amplification oligomer configured to specifically hybridizemiR-221, and a universal reverse primer targets a complement of theextension nucleic acid sequence of the first amplification oligomer. Insome aspects, the amplification reaction comprises a first amplificationoligomer configured to specifically hybridize miR-221 and whichcomprises an extension nucleic acid sequence that is substantiallyidentical to SEQ ID NO:49, and a reverse primer targets a complement ofthe extension nucleic acid sequence of the first amplification oligomer.In some aspects, the amplification reaction comprises a firstamplification oligomer configured to specifically hybridize miR-221 andwhich comprise an extension nucleic acid sequence that is substantiallyidentical to SEQ ID NO:49, and the reverse primer is SEQ ID NO:20. Insome aspects, the amplification reaction comprises a first amplificationoligomer configured to specifically hybridize miR-182, and a universalreverse primer targets a complement of the extension nucleic acidsequence of the first amplification oligomer. In some aspects, theamplification reaction comprises a first amplification oligomerconfigured to specifically hybridize miR-182 and which comprises anextension nucleic acid sequence that is substantially identical to SEQID NO:49, and a reverse primer targets a complement of the extensionnucleic acid sequence of the first amplification oligomer. In someaspects, the amplification reaction comprises a first amplificationoligomer configured to specifically hybridize miR-182 and which comprisean extension nucleic acid sequence that is substantially identical toSEQ ID NO:49, and the reverse primer is SEQ ID NO:20. In some aspects,the amplification reaction is a multiplex amplification comprising atleast two first amplification oligomers, each configured to specificallyhybridize a different target nucleic acid. In some aspects, theamplification reaction is a multiplex amplification comprising at leasttwo first amplification oligomers, each configured to specificallyhybridize a different target nucleic acid, and a reverse primer targetsa complement of the extension nucleic acid sequence of each firstamplification oligomer. In some aspects, the amplification reaction is amultiplex amplification comprising at least two first amplificationoligomers, each configured to specifically hybridize a different targetnucleic acid, and a universal reverse primer targets a complement of theextension nucleic acid sequence of each first amplification oligomer. Insome aspects, the amplification reaction is a multiplex amplificationcomprising at least two first amplification oligomers, each configuredto specifically hybridize a different target nucleic acid and comprisean extension nucleic acid sequence that is substantially identical toSEQ ID NO:49, and a reverse primer targets a complement of the extensionnucleic acid sequence of each first amplification oligomer. In someaspects, the amplification reaction is a multiplex amplificationcomprising at least two first amplification oligomers, each configuredto specifically hybridize a different target nucleic acid and comprisean extension nucleic acid sequence that is substantially identical toSEQ ID NO:49, and the reverse primer is SEQ ID NO:20. In some aspects,the amplification reaction is a multiplex amplification comprising atleast two first amplification oligomers, one of which is configured tospecifically hybridize miR-221, wherein the reverse primer targets acomplement of the extension nucleic acid sequence of a firstamplification oligomer or the reverse primer is a universal reverseprimer that targets a complement of the extension nucleic acid sequenceof each first amplification oligomer. The extension nucleic acidsequence can be substantially identical to SEQ ID NO:49. The reverseprimer can be substantially identical to SEQ ID NO:20. In some aspects,the amplification reaction is a multiplex amplification comprising atleast two first amplification oligomers, one of which is configured tospecifically hybridize miR-182, wherein the reverse primer targets acomplement of the extension nucleic acid sequence of a firstamplification oligomer or the reverse primer is a universal reverseprimer that targets a complement of the extension nucleic acid sequenceof each first amplification oligomer. The extension nucleic acidsequence can be substantially identical to SEQ ID NO:49. The reverseprimer can be substantially identical to SEQ ID NO:20. In some aspects,the amplification reaction is a multiplex amplification comprising atleast two first amplification oligomers, one of which is configured tospecifically hybridize miR-221 and one of which is configured tohybridize to miR-182, wherein a reverse primer targets a complement ofthe extension nucleic acid sequence of one of the first amplificationoligomer and another reverse primer targets a complement of theextension nucleic acid sequence of the other first amplificationoligomer, or wherein the reverse primer is a universal reverse primerthat targets a complement of the extension nucleic acid sequence of eachfirst amplification oligomer. The extension nucleic acid sequence can besubstantially identical to SEQ ID NO:49. The reverse primer can besubstantially identical to SEQ ID NO:20.

In some aspects of the methods, an amplification reaction comprises afirst amplification oligomer that is an extender primer comprising a 3′target hybridizing sequence and a 5′ extension nucleic acid sequence,wherein the 3′ target hybridizing sequence is substantiallycomplementary to about 9 or more contiguous nucleic acids at the 3′ endof the miR-221 target nucleic acid, and further comprises providing apromoter based amplification oligomer comprising a 3′ target hybridizingsequence and a 5′ promoter sequence, wherein the target hybridizingsequence is substantially identical to 17 or more contiguous nucleicacids on 5′ end of the miR-221 target nucleic acid. In some aspects, anamplification reaction comprises a first amplification oligomer forspecifically hybridizing to a miR-221 target nucleic acid that is anextender primer comprising a 3′ target hybridizing sequence and a 5′extension nucleic acid sequence, wherein the 3′ target hybridizingsequence is substantially complementary to about 9 or more contiguousnucleic acids at the 3′ end of the miR-221 target nucleic acid, and afirst amplification oligomer for specifically hybridizing to a miR-182target nucleic acid that is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially complementary toabout 9 or more contiguous nucleic acids at the 3′ end of the miR-182target nucleic acid, and further comprises providing a promoter basedamplification oligomer for hybridizing to a cDNA strand of a miR-221target nucleic acid, the promoter based amplification oligomercomprising a 3′ target hybridizing sequence and a 5′ promoter sequence,wherein the target hybridizing sequence is substantially identical to 12or more contiguous nucleic acids on 5′ end of the miR-221 target nucleicacid, and a promoter based amplification oligomer for hybridizing to acDNA strand of a miR-182 target nucleic acid , the promoter basedamplification oligomer comprising a 3′ target hybridizing sequence and a5′ promoter sequence, wherein the target hybridizing sequence issubstantially identical to 12 or more contiguous nucleic acids on 5′ endof the miR-182 target nucleic acid. In some aspects the amplificationreaction comprises one or more target capture probes. In some aspects,the amplification reaction comprises a target capture probe configuredto capture a miR-221 target nucleic acid. In some aspects theamplification reaction comprises one or more target capture probes. Insome aspects, the amplification reaction comprises a target captureprobe configured to capture a miR-221 target nucleic acid comprising atarget hybridizing sequence that is substantially complementary to SEQID NO:44. In some aspects, the amplification reaction comprises a targetcapture probe that is substantially identical to SEQ ID NO:24. In someaspects, the amplification reaction comprises a target capture probeconfigured to capture a miR-182 target nucleic acid. In some aspects theamplification reaction comprises one or more target capture probes. Insome aspects, the amplification reaction comprises a target captureprobe configured to capture a miR-182 target nucleic acid comprising atarget hybridizing sequence that is substantially complementary to SEQID NO:41. In some aspects, the amplification reaction comprises a targetcapture probe that is substantially identical to SEQ ID NO:23. In someaspects, the amplification reaction is performed in the presence oftarget capture probe and wherein the amplification reaction conditionsare configured to maintain the target capture probe in a hairpinformation. In some aspects, the amplification reaction is an isothermalamplification reaction.

In some aspects of the methods, amplification products are detectedusing a detection probe oligomer. In some aspects of the methods,amplification products are detected using a detection probe oligomerthat target a portion of an amplification product corresponding to thetarget nucleic acid. In some aspects of the methods, amplificationproducts are detected using a detection probe oligomer that target aportion of an amplification product corresponding to the extensionnucleic acid sequence. In some aspects, the detection probe oligomer isa linear detection probe. In some aspects, the detection probe oligomeris a hairpin detection probe. In some aspects, the detection probeoligomer is labeled. In some aspects, the detection probe oligomer isdual labeled. In some aspects, the detection of the target nucleic acidis a real-time detection. In some aspects, the detection probe oligomeris a linear detection probe. In some aspects, the detection probeoligomer is a hairpin detection probe. In some aspects, the detectionprobe oligomer is labeled. In some aspects, the detection probe oligomeris dual labeled. In some aspects, the detection of the target nucleicacid is a real-time detection.

In some aspects, the method are useful for diagnosing cancer, providinga prognosis relating to a cancer, monitoring the effectiveness of acancer treatment, or a combination thereof. In some aspects, the canceris prostrate cancer.

One embodiment is a composition comprising any one or more of theoligomers described herein. Some aspects provide a target capture probethat forms under hybridizing conditions at least a partiallydouble-stranded structure that is a hairpin structure made up of acontiguous linear sequence that includes an internal target hybridizingsequence that is substantially complementary to all or a portion of SEQID NOS:44, 41, 47, 38, 29 or 34 and that forms a single-stranded loopportion of the hairpin structure, wherein the internal targethybridizing sequence is flanked by a capture region and by a terminalregion that binds to the capture region to form a double-stranded stemportion of the hairpin structure and wherein the capture region furthercomprises a substantially homopolymeric nucleic acid sequence and theterminal region further comprises a substantially homopolymeric nucleicacid sequence that is complementary to the capture region sequence. Insome aspects, the capture region sequence is selected from the groupconsisting of dT.sub.0-3A.sub.10-30. In some aspects, the capture probeis at least 95% identical to SEQ lD NOS:22, 23, 24, 25 or 26.

One embodiment provides an amplification reaction mixture comprising anyone or more of the oligomers described herein. In one aspect, theamplification reaction mix comprises any one or more of, the targetcapture oligomers described herein. In one aspect, the amplificationreaction mix comprises any one or more of the first amplificationoligomers described herein. In one aspect, the amplification reactionmix comprises any one or more of the promoter-based amplificationoligomers described herein. In one aspect, the amplification reactionmix comprises any one or more of the reverse primer oligomers describedherein. In one aspect, the amplification reaction mix comprises any oneor more of the detection probe oligomers described herein. In oneaspect, the amplification reaction mix comprises any one or more of thetarget capture oligomers described herein, first amplification oligomersdescribed herein, promoter based oligomers described herein, reverseprimer oligomers described herein and detection probe oligomersdescribed herein. In one aspect there is provided an amplificationreaction mixture comprising a small RNA, miR21, miR-34b, miR-182,miR-221, miR-222 or miR-802 target capture probe that forms underhybridizing conditions at least a partially double-stranded structurethat is a hairpin structure made up of a contiguous linear sequence thatincludes an internal target hybridizing sequence that is substantiallycomplementary to all or a portion of small RNA, miR21, miR-34b, miR-182,miR-221, miR-222 or miR-802 and that forms a single-stranded loopportion of the hairpin structure, wherein the internal targethybridizing sequence is flanked by a capture region and by a terminalregion that binds to the capture region to form a double-stranded stemportion of the hairpin structure and wherein the capture region furthercomprises a substantially homopolymeric nucleic acid sequence and theterminal region further comprises a substantially homopolymeric nucleicacid sequence that is complementary to the capture region sequence; anda first amplification oligomer configured to hybridize to a small RNA,miR21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleic acidthat is an extender primer comprising a 3′ target hybridizing sequenceand a 5′ extension nucleic acid sequence, wherein the 3′ targethybridizing sequence is substantially identical to about 9 or morecontiguous nucleic acids at the 5′ end of the target hybridizingsequence of the target capture probe. In one aspect there is provided anamplification reaction mixture comprising a miR21, miR-34b, miR-182,miR-221, miR-222 or miR-802 target capture probe that forms underhybridizing conditions at least a partially double-stranded structurethat is a hairpin structure made up of a contiguous linear sequence thatincludes an internal target hybridizing sequence that is substantiallycomplementary to all or a portion of SEQ ID NOS:38, 29, 41, 44, 47 or 34and that forms a single-stranded loop portion of the hairpin structure,wherein the internal target hybridizing sequence is flanked by a captureregion and by a terminal region that binds to the capture region to forma double-stranded stem portion of the hairpin structure and wherein thecapture region further comprises a substantially homopolymeric nucleicacid sequence and the terminal region further comprises a substantiallyhomopolymeric nucleic acid sequence that is complementary to the captureregion sequence; and a first amplification oligomer configured tohybridize to a small RNA, miR21, miR-34b, miR-182, miR-221, miR-222 ormiR-802 target nucleic acid that is an extender primer comprising a 3′target hybridizing sequence and a 5′ extension nucleic acid sequence,wherein the 3′ target hybridizing sequence is substantially identical toabout 9 or more contiguous nucleic acids at the 5′ end of the targethybridizing sequence of the target capture probe. In one aspect there isprovided an amplification reaction mixture comprising a miR-221 targetcapture probe that forms under hybridizing conditions at least apartially double-stranded structure that is a hairpin structure made upof a contiguous linear sequence that includes an internal targethybridizing sequence that is substantially complementary to all or aportion of SEQ ID NO:44 and that forms a single-stranded loop portion ofthe hairpin structure, wherein the internal target hybridizing sequenceis flanked by a capture region and by a terminal region that binds tothe capture region to form a double-stranded stem portion of the hairpinstructure and wherein the capture region further comprises asubstantially homopolymeric nucleic acid sequence and the terminalregion further comprises a substantially homopolymeric nucleic acidsequence that is complementary to the capture region sequence; and afirst amplification oligomer configured to hybridize to a miR-221 targetnucleic acid that is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially identical to about 9or more contiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-221 target capture probe. In one aspect there isprovided an amplification reaction mixture for multiplex amplificationscomprising (a) a miR-221 target capture probe that forms underhybridizing conditions at least a partially double-stranded structurethat is a hairpin structure made up of a contiguous linear sequence thatincludes an internal target hybridizing sequence that is substantiallycomplementary to all or a portion of SEQ ID NO:44 and that forms asingle-stranded loop portion of the hairpin structure, wherein theinternal target hybridizing sequence is flanked by a capture region andby a terminal region that binds to the capture region to form adouble-stranded stem portion of the hairpin structure and wherein thecapture region further comprises a substantially homopolymeric nucleicacid sequence and the terminal region further comprises a substantiallyhomopolymeric nucleic acid sequence that is complementary to the captureregion sequence and further comprising a miR-182 target capture probethat forms under hybridizing conditions at least a partiallydouble-stranded structure that is a hairpin structure made up of acontiguous linear sequence that includes an internal target hybridizingsequence that is substantially complementary to all or a portion of SEQID NO:41 and that forms a single-stranded loop portion of the hairpinstructure, wherein the internal target hybridizing sequence is flankedby a capture region and by a terminal region that binds to the captureregion to form a double-stranded stem portion of the hairpin structureand wherein the capture region further comprises a substantiallyhomopolymeric nucleic acid sequence and the terminal region furthercomprises a substantially homopolymeric nucleic acid sequence that iscomplementary to the capture region sequence; and (b) a firstamplification oligomer configured to hybridize to a miR-221 targetnucleic acid that is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially identical to about 9or more contiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-221 target capture probe, and further comprising afirst amplification oligomer configured to hybridize to a miR-182 targetnucleic acid that is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially identical to about 9or more contiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-182 target capture probe. In one aspect there isprovided an amplification reaction mixture for multiplex amplificationscomprising (a) two or more target capture probes configured tospecifically hybridize to two or more of a small RNA, miR21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acid, wherein thetarget capture oligomers form under hybridizing conditions at least apartially double-stranded structure that is a hairpin structure made upof a contiguous linear sequence that includes an internal targethybridizing sequence that is substantially complementary to all or aportion of each of their target nucleic acids and that form asingle-stranded loop portion of the hairpin structure, wherein theinternal target hybridizing sequences are flanked by capture regions andby terminal regions that binds to the capture regions to formdouble-stranded stem portion of the hairpin structure and wherein thecapture regions further comprises a substantially homopolymeric nucleicacid sequence and the terminal regions further comprise substantiallyhomopolymeric nucleic acid sequence that is complementary to the captureregion sequences; and (b) two or more first amplification oligomersconfigured to hybridize two or more of a small RNA, miR21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acids, wherein thefirst amplification oligomers are extender primers comprising 3′ targethybridizing sequences and 5′ extension nucleic acid sequences, whereinthe 3′ target hybridizing sequences are substantially identical to about9 or more contiguous nucleic acids at the 5′ end of the targethybridizing sequence of the corresponding target capture probes.

In some aspects the amplification reaction mixture further comprises apromoter based oligomer that comprises a target hybridizing sequencethat is substantially complementary to 12 or more contiguous nucleicacids on 3′ end of the miR-221 target capture probe. In some aspects,the promoter-based oligomer is at least 95% identical to SEQ ID NO:11.In some aspects, the promoter-based oligomer is SEQ ID NO:11. In someaspects the amplification reaction mixture further comprises a promoterbased oligomer that comprises a target hybridizing sequence that issubstantially complementary to 12 or more contiguous nucleic acids on 3′end of the miR-182 target capture probe. In some aspects, thepromoter-based oligomer is at least 95% identical to SEQ ID NO:10. Insome aspects, the promoter-based oligomer is SEQ ID NO:10. In someaspects, the miR-221 target capture probe is at least 95% identical toSEQ ID NO:24. In some aspects, the miR-221 target capture probe is SEQID NO:24. In some aspects, the miR-182 target capture probe is at least95% identical to SEQ ID NO:23. In some aspects the miR-182 targetcapture probe is SEQ ID NO:23. In some aspects, the miR-221 extenderprimer is at least 95% identical to SEQ ID NO:17. In some aspects, themiR-221 extender primer is SEQ ID NO:17. In some aspects the miR-182extender primer is at least 95% identical to SEQ ID NO:16. In someaspects the miR-182 extender primer is SEQ ID NO:16. In some aspects,the amplification reaction mixture further comprises a reverse primer.In some aspects, the reverse primer targets a portion of a sequence thatis complementary to the extension nucleic acid sequence of an extenderprimer. In some aspects, the reverse primer is SEQ ID NO:20. in someaspects, the target capture probe comprises at least one 2′-OMe residuein the target hybridizing sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an embodiment showing capture and detection of atarget nucleic acid by mixing target nucleic acid (shown as the sequenced c b a) with a hairpin capture probe which has complementary sequencesat its 5′ and 3′ ends (shown as poly-T and poly-A regions) flanking atarget complementary region (shown as the sequence a′ b′ c′ d′ and alsoreferred to as a target hybridizing sequence) to form a capture hybridmade up of the target nucleic acid hybridized to target hybridizingregion of the opened capture probe, and a portion of a capture probe(poly-A region) hybridized to a complementary immobilized probe (shownas poly-T attached to a capture support), followed by releasing thetarget into solution where it forms a detection hybrid made up of adetection probe (shown by the sequence d′ c′ b′ a′) hybridized to thetarget nucleic acid to produce a detectable signal (shown by astar-shaped character) to indicate the presence of the target. Anamplification step is not shown in this Figure. Detection can beperformed directly on isolated target nucleic acids or an amplificationstep can be included and detection performed in real-time or endpoint,

FIG. 2 illustrates an embodiment showing capture and detection of atarget nucleic acid that mixes the target nucleic acid (shown as thesequence a b c d e) with a completely or partially double-strandedcapture probe that contains complementary sequences on the two strands(shown as poly-A and poly-T sequences) and one target-complementaryregion (shown as the sequence a′ b′ c′ on the poly-T containing strandand also referred to as a target hybridizing sequence), to form acapture hybrid made up of the target nucleic acid hybridized to thetarget hybridizing region of the capture probe strand, and anotherportion of the capture probe strand (poly-T) hybridized to acomplementary immobilized probe (shown as poly-A attached to a capturesupport), followed by releasing the target nucleic acid into solution toform a detection hybrid made up of a detection probe (shown by thesequence a′ b′ c′ d′ e′) hybridized to the target nucleic acid toproduce a detectable signal (shown by a star-shaped character) toindicate the presence of the target. An amplification step is not shownin this Figure. Detection can be performed directly on isolated targetnucleic acids or an amplification step can be included and detectionperformed in real-time or endpoint.

FIG. 3 illustrates a general schematic of miRNA TMA amplificationreaction using an extender primer oligomer, a promoter based oligomerand a reverse primer oligomer.

FIGS. 4 a-d shows the specificity of the miRNA TMA assay. A) Sequencesof mature miRNAs tested. B) Amplification curves from tests of themiR-802 assay on various input copy numbers of synthetic miRNA targets.RFU: Relative fluorescence units. C) Calibration chart for theamplification curves in B. D) Calibration chart for the miR-802 assaytested on various copy number input of mature target vs. pre-miR-802transcript,

FIGS. 5 a-c shows the specificity of the miR TMA assay. A) Sequences ofmature miRNAs tested. B) Amplification curves from tests of the miR-34bassay on various copy number input of related miR-34 family membersalong with unrelated let-7d non-target nucleic acids. RFU: Relativefluorescence units. C) Calibration chart for the amplification curves inB.

FIGS. 6 a-b shows the specificity of the miR TMA assay. A)Template-dependent amplification of prostate cancer candidate miRNAs.Amplification reactions were run in triplicate (without target capture).The slope and R.sup.2 values were determined from log copy points 1.7 to7.7 (5 to 5×10⁷ copies per reaction). B) Specificity test of the miR-221assay for miR-221 target nucleic acid as compared to miR-222, miR-30b,and miR-802 related and unrelated non-target nucleic acids.

FIGS. 7 a-d shows miRNA assays with target capture. A) Capture anddetection of known inputs of synthetic miR-221 added directly to STMbuffer or to STM containing 10 ng of total cellular RNA (derived fromVCaP or LNCaP cells, as indicated). Control reactions were run withsynthetic miR-221 spiked directly into amplification reagent (withouttarget capture or total RNA). B) miR-221 copy levels determined by acommercial TaqMan RT-qPCR assay. C) miR-221 copy levels determined by amiR real-time TMA assay (with target capture). D) miR-221 copy numbersdetermined in prostate cancer xenografts using a commercial TaqManRT-qPCR assay (without target capture) and a miR real-time TMA assay(with target capture). For all real-time TMA samples, 10 ng total RNAwas assayed per reaction in triplicate.

FIGS. 8 a-b shows an absolute copy number determination for A) miR-21and B) miR-221, miR-222, and miR-182. Synthetic mature microRNAs werediluted 10-fold from 1 x 10.sup.9 copies per reaction for constructionof each standard curve. For each reverse transcription reaction, 10 ngof total RNA was used and reactions were run in triplicate according tomanufacturer's instructions.

DETAILED DESCRIPTION

This invention is useful for detecting a target nucleic acid of interestpresent in a sample. The small RNA target nucleic acid is preferably amicro RNA, miR-21, miR-34b, miR-182, miR-221, miR-222 or miR-802. Themethods use relatively few and easily performed steps to isolate and/orconcentrate the target nucleic acid from other sample components and todetect the target nucleic acid. Samples are typically one or more cellsobtained as a specimen that can be used to determine the presence orabundance of a target nucleic acid that is a biomarker for a disorder.For example, the sample can be one or more blood cells that can be usedfor determining a disorder wherein a biomarker is present in bloodcells. Another example, the sample can be one or more biopsied cellsthat can be used for determining the presence of a disorder wherein abiomarker is present in the tissue or organ from which the biopsy wastaken. The methods include isolating the target nucleic acid from thesample. In some instances the target nucleic acid is released from acell by lysing the cell in which the target nucleic acid is suspected ofbeing present. In some instances, the released target nucleic acid isthen isolated away from other components of the sample, which mayinclude cellular debris is the target nucleic acid was released by celllysis. Isolation can include general nucleic acid isolation, which canbe done using kits like the mirVana kits (ABI, Foster City, Calif.);Trizol LS (Invitrogen, Carlsbad, Calif.); Micro RNA Isolation kits(Stratagene, La Jolla, Calif.) and High-Pure miRNA Isolation kits(Roche, Indianapolis, Ind.). Preferably, isolation of the target nucleicacid(s) include a capture step in which a target nucleic acid iscaptured by using one or more target capture probes to form a capturehybrid that is linked to a capture support. The capture hybrid is thenseparated from other sample components including non-target nucleicacids. Captured target nucleic acids are then released from the capturesupport and assayed by one or more downstream methodologies, such as anamplification reaction and/or a detection reaction. The target nucleicacids can be captured directly from sample cell lysates or can becaptured from isolated nucleic acids, such as a total RNA isolation or afractionated population of RNA such as a population of small RNA.

Removal of non-target nucleic acids is beneficial in that non-targetnucleic acids potentially negatively impact the detection of small RNAthrough non-specifically hybridizing to assay system oligomers, crowdingthe reaction to make scarce target nucleic acid species undetectable orunreliably detectable and otherwise interfering with the assay. When adetection reaction is performed in the presence of a generalizedpopulation of nucleic acids, e.g., total RNA or size fractionated RNA,the non-target sequences in the reaction can non-specifically hybridizewith the assay oligonucleotides, thereby removing needed reagents fromthe assay. A non-specifically hybridized reagent is not available forits intended target, and furthermore, may be used by the non-specifictarget for generating false positive signals. The target capture stepcan be performed in place of a general nucleic acid isolation procedure,or can be performed on the nucleic acid recovered from such a generalisolation. Preferably, in a downstream assay the target nucleic acid isdetected. Preferably, the detection step is a nucleic acid probe baseddetection step. The detection step is sometime preceded by anamplification step; though this is not a requirement.

Target nucleic acids are typically from about 17-27 nucleotides inlength. When a target nucleic acid is captured and then detected, thetarget capture oligomer and the detection probe oligomer comprise targethybridizing sequences that are substantially similar to one another.Thus, these two oligomers have target hybridizing sequences that competefor the target nucleic acid. In one embodiment, the target captureoligomer is configured so that under a first set of conditions thetarget capture oligomer is not in a hairpin form, and thus the targethybridizing sequence is available to hybridize to a target nucleic acid.However, under a second set of conditions, the target capture oligomeris in a hairpin form. The detection probe, on the other hand, isconfigured to hybridize to the target nucleic acid under this second setof conditions. The target capture probe is therefore configured to beunavailable to hybridize the small target nucleic acid under theconditions wherein the detection probe is present for hybridizing thetarget. In a method, one or more target nucleic acids are captured froma sample or from a general nucleic acid isolate under a first set ofconditions and isolated away from the remaining debris and/or non-targetnucleic acids. The one or more target nucleic acids along with theirrespective target capture oligomers are released into solution andcombined with one or more detection probe oligomers specific for each ofthe target nucleic acids. The solution at this point contains targetnucleic acid(s), target capture oligomer(s) and detection probeoligomer(s), wherein the target hybridizing sequences of a targetcapture oligomer and a detection probe oligomer configured to hybridizea target nucleic acid are substantially identical. A set of conditionsis then applied to the solution so that the detection probe oligomerpreferentially hybridizes to the target nucleic acid, not the targetcapture oligomer. When a target nucleic acid is captured, amplified andthen detected, the target capture oligomer and the amplificationoligomers comprise target hybridizing sequences that are substantiallysimilar or substantially identical to one another. Thus, these oligomershave target hybridizing sequences that compete for the target nucleicacid or that can hybridize together. In one embodiment, the targetcapture oligomer is configured so that under a first set of conditionsthe target capture oligomer is not in a hairpin form, and thus thetarget hybridizing sequence is available to hybridize to a targetnucleic acid. However, under a second set of conditions, the targetcapture oligomer is in a hairpin form. The amplification oligomers, onthe other hand, are configured to hybridize to the target nucleic acid,or complement thereof under this second set of conditions. The targetcapture probe is therefore configured to be unavailable to hybridize thesmall target nucleic acid under the conditions wherein the amplificationoligomers are present for hybridizing the target or its complement. In amethod, one or more target nucleic acids are captured from a sample orfrom a general nucleic acid isolate under a first set of conditions andisolated away from the remaining debris and/or non-target nucleic acids.The one or more target nucleic acids along with their respective targetcapture oligomers are released into solution and combined withamplification oligomers specific for each of the target nucleic acids.The solution at this point contains target nucleic acid(s), targetcapture oligomer(s) and amplification oligomers(s), wherein the targethybridizing sequences of a target capture oligomer and a firstamplification oligomer are configured to hybridize all or a portion ofthe same target sequence on a target nucleic acid, and wherein all or aportion of the second amplification oligomer target hybridizing sequenceis substantially identical to all or a portion of the target captureoligomer target hybridizing sequence. A set of conditions is thenapplied to the solution so that the amplification oligomerspreferentially hybridize to the target nucleic acid, not the targetcapture oligomer, and amplification products are generated.

These methods are useful for detecting the presence or absence of one ormore target nucleic acids in a sample. These methods are useful fordetecting the abundance (including expression level) of one or moretarget nucleic acids in a sample. These methods are useful particularlyfor detecting small target nucleic acids that may be present at diluteconcentrations in a sample, e.g., a small nucleic acids excreted inurine or present in a cellular or tissue extract. These methods are alsouseful for assaying many samples, preferably simultaneously or in rapidsuccession, such as in an automated high through-put system because thecapture and detection steps can be performed in a single reactionchamber per sample. In addition to directly detecting one or more targetnucleic acids present in a sample, the methods herein further include anucleic acid amplification wherein the target nucleic acid(s) areamplified and the amplification product(s) are detected. Preferably thetarget nucleic acids are isolated away from cellular debris and/ornon-target nucleic acids. Isolated target nucleic acids are moreaccurately amplified and detected because interference by a non-targetnucleic acid is reduced. Capture and amplification of one or more targetnucleic acids uses a target capture oligomer and at least a firstamplification oligomer. The target capture oligomer and the firstamplification oligomer, therefore, are competing for hybridization to asmall target sequence. Additional amplification oligomers include apromoter based amplification oligomer, a reverse primer oligomer orboth. The target capture oligomer and the promoter-based oligomer canhybridize together. Thus, it is preferably that the target captureoligomer is subjected to chemical and/or physical conditions that areconfigured to make the target capture oligomer unavailable to interferewith the amplification reaction. Preferably, the unavailable targetcapture oligomer is in a double stranded or partially double strandedstructure. Preferably, the unavailable target capture oligomer is ahairpin structure.

Amplification methods include PCR (RT or qRT), transcription mediatedamplification, or other amplification methods. Preferably, theamplification method is a transcription mediated amplification methodand the amplification oligomers include a first primer oligomerconfigured to hybridize to at least a portion of a target nucleic acidand to further add an extension nucleic acid sequence to the targetnucleic acid, a promoter based amplification oligomer configured tohybridize to a cDNA strand section complementary to the target nucleicacid and a reverse primer oligomer configured to hybridize to a portionof the extension sequence. In a multiplex reaction for detecting two ormore target nucleic acids, the first primer oligomer preferably adds thesame extension nucleic acid sequence to each target nucleic acid, inwhich case the reverse primer oligomer is a universal primer oligomer.Preferably, the methods for detecting the presence or abundance of atarget nucleic acid include an amplification reaction that takes placein the presence of a target capture oligomer. Preferably theamplification reaction is configured to amplify one or more targetnucleic acids in the presence of one or more target capture oligomerswithout interference from the target capture oligomer. Preferably theone or more target capture oligomers are configured to form a hairpinstructure under conditions wherein the target nucleic acid is releasedand to remain in a hairpin configuration during an amplificationreaction. Preferably the one or more target capture oligomers areconfigured to form a hairpin structure under conditions wherein thetarget nucleic acid is released and to remain in a hairpin configurationduring transcription mediated amplification reaction.

The presence and amount of one or more target nucleic acids in a sampleare accurately determined using the oligomers and methods of thiscurrent disclosure. Target nucleic acids can be biomarkers fordetermining a disease state, determining a prognosis, developing a drugcompound or monitoring the efficacy of a treatment. The presence of orthe relative abundance of biomarkers is useful in such efforts. However,the value of information received from a biomarker detection assaydiminishes if the results are negatively impacted by contaminations,including non-target nucleic acids. Some problems with non-targetnucleic acids include non-specific hybridization of the oligomerreagents to the non-target nucleic acids, thereby depleting reagentsand/or providing non-target amplification products as false positivesignals. Additionally, the presence of cellular debris and non-targetnucleic acids can bury small and less abundant target nucleic acids sothat the oligomer reagents cannot hybridize with their targets, leadingto false positives. Thus, it is preferred that the detection methods fordetermining the presence or abundance of a target nucleic acid include astep for isolating the target nucleic acids away from interferingcontamination.

To better understand the various embodiments of the invention, some ofthe terms used in the description of the invention are more fullydescribed below.

A “Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine, 5-methylisocytosine, isoguanine; TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed.,1992, Abraham et al., 2007, BioTechniques 43: 617-24), which includederivatives of purine or pyrimidine bases (e.g., N4-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O.sup.6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O.sup.4-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” residues in whichthe backbone does not include a nitrogenous base for one or moreresidues (U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′ methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., 2004, Biochemistry 43(42):13233-41). Nucleic acids may includemodified bases to alter the function or behavior of the nucleic acid,e.g., addition of a 3′-terminal dideoxynucleotide to block additionalnucleotides from being added to the nucleic acid. Synthetic methods formaking nucleic acids in vitro are well known in the art although nucleicacids may be purified from natural sources using routine techniques.

A nucleic acid “backbone” refers to groups or linkages known in the art(Eschenmoser, 1999, Science 284:2118-2124), such as, e.g.,sugar-phosphodiester linkages, 2′-O-methyl linkages, guanidine linkersin DNA (“DNG”), S-methylthiourea linkers, methylphosphonate linkages,phosphoramidite linkages, amide backbone modifications as in polyamideor peptide nucleic acids (PNA), phosphorothioate linkages, phosphonicester nucleic acid linkages, pyranosyl oligonucleotide linkages,bicyclo- and tricyclo-nucleic acid linkages, formacetal and3′-thioformacetal linkages, morpholino linkages, or other modificationsof the natural phosphodiester internucleoside bond, or combinations ofsuch linkages in a single backbone (Majlessi et al., 1998, Nucl. AcidsRes. 26(9):2224-2229; Dempcy et al., 1995, Proc. Natl. Acad. Sci. USA92:6097-6101; Browne et al., 1995, Proc. Natl. Acad. Sci. USA92:7051-7055; Arya & Bruice, 1998, J. Am. Chem. Soc. 120:6619-6620;Reynolds et al., 1996, Nucl. Acids Res. 24(22):4584-4591; Gryaznov &Chen, 1994, Am. Chem. Soc. 116:3143-3144; Chaturvedi et al., 1996, Nucl.Acids Res. 24(12):2318-2323; Hyrup & Nielsen, 1996, Bioorg. & Med. Chem.4:5-23; Hydig-Hielsen et al., PCT App. No. WO 95/32305; Mesmaeker etal., Syn. Lett., November 1997:1287-1290; Peyman et al., 1996, Angew.Chem. Int. Ed. Engl. 35(22):2636-2638; Aerschot et al., 1995, Angew.Chem. Int. Ed. Engl. 34(12):1338-1339; Koshkin et al. 1998, J. Am. Chem.Soc. 120:13252-13253; Steffens & Leumann, 1997, J. Am. Chem. Soc.119:11548-11549; Jones et al., 1993, J. Org. Chem. 58:2983-2991;Summerton & Weller, 1997, Antisense & Nucl. Acid Drug Dev. 7:187-195;Stirchak et al., 1989, Nucl. Acids Res. 17(15):6129-6141). A nucleicacid backbone may include a mixture of linkages in the same nucleic acid(e.g., sugar-phosphodiester and phosphorothioate) or may have all of onetype of linkages (e.g., all amide modification linkages in an oligomer).

The interchangeable terms “oligomer,” “oligo” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of about 5nt residues and an upper limit of about 500 to 900 nt residues. In someembodiments, oligonucleotides are in a size range having a lower limitof about 12 to 15 nt and an upper limit of about 50 to 600 nt, and otherembodiments are in a range having a lower limit of about 15 to 20 nt andan upper limit of about 22 to 100 nt. Oligonucleotides may be purifiedfrom naturally occurring sources, but or may be synthesized using any ofa variety of well known enzymatic or chemical methods. The termoligonucleotide does not denote any particular function to the reagent;rather, it is used generically to cover all such reagents describedherein. An oligonucleotide may serve various different functions. Forexample, it may function as a primer if it is specific for and capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase, it may provide a promoter ifit contains a sequence recognized by an RNA polymerase and allows fortranscription (e.g., a promoter-based oligomer), and it may function toprevent hybridization or impede primer extension if appropriatelysituated and/or modified.

A “target” or “target sequence” or “target nucleic acid” refers to asequence of nucleotide bases present in a nucleic acid, or portion of anucleic acid, to which another sequence binds, e.g., by using standardcomplementary base pairing. Preferably, herein, a target nucleic acid isa small RNA having a length of from about 17 to about 27 contiguousnucleotides. Small RNA target nucleic acids include, miRNA, siRNA,shRNA, antisense RNA, snRNA or the like. Those skilled in the art willappreciate that a target nucleic acid may exist in different forms,i.e., single-stranded, double-stranded, triple-stranded, or mixturesthereof, such as in a partially double-stranded hairpin structure orpartially double-stranded duplex structure, and will further appreciatethat a target sequence may be present on any strand (+ or −) of thestructure. It is well known in the art that a multi-stranded nucleicacid is readily converted to its single-strand components by usingstandard methods, such as by heating a nucleic acid above its meltingtemperature (Tm) and/or by using chemical denaturants.

By “complementary” or “complementarity of' nucleic acids is meant that anucleotide sequence in one strand of nucleic acid, due to orientation ofthe functional groups, will hydrogen bond to another sequence on anopposing nucleic acid strand. The complementary bases typically are, inDNA, A with T and C with G, and, in RNA, C with G, and U with A.“Substantially complementary” means that a sequence in one strand is notcompletely and/or perfectly complementary to a sequence in an opposingstrand, but that sufficient bonding occurs between bases on the twostrands to form a stable hybrid complex in set of hybridizationconditions (e.g., salt concentration and temperature). Such conditionscan be predicted by using the sequences and standard mathematicalcalculations known to those skilled in the art to predict the Tm ofhybridized strands, or by empirical determination of Tm by using routinemethods. Tm refers to the temperature at which a population ofhybridization complexes formed between two nucleic acid strands are 50%denatured. At a temperature below the Tm, formation of a hybridizationcomplex is favored, whereas at a temperature above the Tin, melting orseparation of the strands in the hybridization complex is favored. Tmmay be estimated for a nucleic acid having a known G+C content in anaqueous 1 M NaCl solution by using, e.g., Tm=81.5+0.41(% G+C), althoughother Tm computations are known in the art which take into accountnucleic acid structural characteristics. Nucleic acid sequences areidentical when their contiguous nucleotide arrangements are the same.Identical sequences includes those that have a modified residue in onesequence, but not the other, so long as the residue is basically thesame (e.g., a 2′-OMe residue in one sequence is still identical to astrand lacking the TOMe modification). Substantially identical sequencesare those that contain sequence differences between the two strands, butthe strands retain similar hybridization properties. Identity andsubstantial identity between sequences are understood and easilydetermined by ordinarily skilled artisans. Sequences herein that are atleast a certain percent identical or complementary to another sequence,means that the sequences includes all rational numbers from thereferenced percent identity to 100%. For example, at least 80% means allnatural number percentages 80, 81, 82, 82, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 and 100, as well as any fraction inbetween (e.g., 82.6, 91.1, 97.9, etc). Ordinarily skilled artisans candetermine percent complementarity and percent identity.

“Hybridization condition” refers to the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions and hydrogen bonding to produce ahybridization complex. Such conditions include the chemical componentsand their concentrations (e.g., salts, chelating agents, formamide) ofan aqueous or organic solution containing the nucleic acids, and thetemperature of the mixture. Other well known factors, such as the lengthof incubation time or reaction chamber dimensions may contribute to theenvironment (e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).

A “label” refers to a molecular moiety that is detectable or produces adetectable response or signal directly or indirectly, e.g., bycatalyzing a reaction that produces a detectable signal. Labels includeluminescent moieties (such as fluorescent, bioluminescent, orchemiluminescent compounds), radioisotopes, members of specific bindingpairs (e.g., biotin and avidin), enzyme or enzyme substrate, reactivegroups, or chromophores, such as a dye or particle that results indetectable color.

A “detection probe” is a oligomer or polymer that binds specifically toa target sequence and which binding results, directly or indirectly, ina detectable signal to indicate the presence of the target sequence. Adetection probe need not be labeled to produce a detectable signal,e.g., an electrical impulse resulting from binding the probe to itstarget sequence may be the detectable signal. A “labeled probe” is aprobe that contains or is linked, directly or indirectly, to a label.Methods of making and/or using labeled probes are well known in the art(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.,Chapt. 10; U.S. Pat. No. 6,361,945, Becker et al.; U.S. Pat. No.5,658,737, Nelson et al.; U.S. Pat. No. 5,656,207, Woodhead et al.; U.S.Pat. No. 5,547,842, Hogan et al.; U.S. Pat. No. 5,283,174, Arnold etal.; U.S. Pat. No. 4,581,333, Kourilsky et al.; U.S. Pat. No. 5,731,148,Becker et al.). For example, detection probes may include anon-nucleotide linker and a chemiluminescent label attached to thelinker (U.S. Pat. Nos. 5,185,439, 5,585,481 and 5,639,604, Arnold etal.).

Examples of detection probes include linear or hairpin oligonucleotidesof about 5 to 50 nucleotides in length having an attached label. Linearprobes include taqman probes and AE probes. Hairpin probes includemolecular torches and molecular beacons. A molecular torch probecomprises a target binding domain and a closing domain, which allow themolecular torch to exist in open and closed conformations, depending onwhether the torch is bound to a target. (See also, U.S. Pat. Nos.6,849,412; 6,835,542; 6,534,274; and 6,361,945). Molecular beacons aregenerally described in Tyagi et al., 1998, Nature Biotechnol. 16:49-53,and in U.S. Pat. Nos. 5,118,801; and 5,312,728. Methods for using suchhairpin probes to detect the presence of a target sequence are wellknown in the art. In some instances, detection probes have a nucleotidesequence that is of the same sense as and comprises a target hybridizingsequence that is substantially identical to the target hybridizingsequence of the capture probe used in the assay. In other instances, thedetection probes are configured to have a target hybridizing sequencethat is substantially complementary to an amplification product, which,if using an extender primer, could comprise all or part of the extenderprimer's target hybridizing sequence or all or part of the extenderprimer's extension sequence.

Labeled detection probes can have an attached chemiluminescent marker,e.g., an acridinium ester (AE) compound (U.S. Pat. Nos. 5,185,439,5,639,604, 5,585,481, and 5,656,744). The acridinium ester label istypically, attached to a central region of the probe near a region of Aand T base pairs by using a non-nucleotide linker which restricts theamines of the nucleotide bases on both sides of the AE and provides asite for intercalation. Alternatively, an AE label may be attached tothe 3′ or 5′ terminus of the detection probe, which is then used inconjunction with a second oligomer that hybridizes adjacent to thedetection probe on the target nucleic acid to restrict the effects ofnearby amine contributed by the target nucleic acid. Another embodimentattaches an AE label at or near the site of a mismatch with a relatednon-target polynucleotide sequence, to permit discrimination between therelated sequence and the target sequence that may differ by only onenucleotide because the area of the duplex around the mismatch site issufficiently destabilized to render the AE on the probe hybridized tothe related non-target sequence susceptible to hydrolysis degradation.Also, labeled probes can be labeled at one end with a fluorescent label(“F”) that absorbs light of a particular wavelength or range and emitslight another emission wavelength or range and at the other end with aquencher (“Q”) that dampens, partially or completely, signal emittedfrom the excited F when Q is in proximity with the fluorophore. Such aprobe may be referred to as labeled with a fluorescent/quencher (F/Q)pair. Further, labeled probes can comprise an AE and an F or an AE and aQ for amplified or quenched signal.

“Homogeneous detectable label” refers to a label whose presence can bedetected in a homogeneous manner depending on whether the label is boundor unbound to a target. A homogeneous detectable label can be detectedin a “homogeneous reaction” without physically separating unbound formsof the label from the mixture before the detection step. It will beappreciated that a homogeneous reaction may occur in solution or on asupport such as a microarray, biochip, or gene chip. Preferredhomogeneous detectable labels and conditions for their detection areknown (U.S. Pat. No. 5,283,174, Arnold et al.; U.S. Pat. No. 5,656,207,Woodhead et al.; U.S. Pat. No. 5,658,737, Nelson et al.).

An “immobilized probe” provides a means for joining a capture hybridcontaining a target nucleic acid to a capture support. A preferredimmobilized probe is a nucleic acid oligomer or polymer joined to asupport, and which binds, directly or indirectly to a target nucleicacid to facilitate separation of the bound target nucleic acid fromunbound material, such as other sample components. In a preferredembodiment, the target nucleic acid is indirectly bound to theimmobilized probe via a target capture probe. Any of a variety ofmaterials may be used as a capture support, e.g., matrices or particlesmade of nitrocellulose, nylon, glass, polyacrylate, mixed polymers,polystyrene, silane polypropylene, and magnetically attractablematerials. Monodisperse magnetic spheres are a preferred embodiment of acapture support because they are relatively uniform in size and readilyretrieved from solution by applying a magnetic force to the reactioncontainer, preferably in an automated system. An immobilized probe maybe linked directly to the capture support, e.g., by using any of avariety of covalent linkages, chelation, or ionic interaction, or may belinked indirectly via one or more linkers joined to the support.

A “capture probe,” “target capture probe” or “ target capture oligomer”provides a means for joining a target nucleic acid and an immobilizedprobe, preferably by hybridization of complementary sequences. A captureprobe comprises a target hybridizing sequence and a binding member forattaching the capture probe, or a hybridization complex that includesthe capture probe, to an immobilized probe. Such binding member may be ahomopolymeric or substantially homopolymeric nucleotide sequence that iscomplementary to a sequence of an immobilized probe, or may be a firstbinding member of specific binding pair, wherein the second bindingmember is attached to a solid support (e.g., biotin and avidin orstreptavidin). In a preferred embodiment, the first binding member is asubstantially homopolymeric nucleotide sequence; more preferably, thesubstantially homopolymeric nucleotide sequence is poly-T.sub.0-3A.sub.5-30, and the second binding member is a sequence substantiallycomplementary to the first such that the first and second bindingmembers hybridize under appropriate conditions. (See e.g., U.S. Pat. No.6,110,678 and PCT Pub. No. WO 06/07567, both of which are incorporatedherein by reference).

Examples of capture probes are oligomers of DNA, RNA and/or analogsthereof that are comprised of target hybridizing sequences of at least10 nucleotides complementary to a target nucleic acid. Target captureoligomers typically comprise a target hybridizing sequence of about 20nucleotides. Target hybridizing regions can include at least one2′-O-methyl residue structure to enhance binding to target nucleicacids. Embodiments of capture probes include oligomers that have atarget hybridizing sequences of about 15 to 25 nucleotides covalentlyattached to a substantially homopolymeric sequence at the 3′ and/or 5′end of the target hybridizing sequence. Some preferred capture probeembodiments include an oligomer that comprises a target hybridizingsequence flanked on both its 3′ end and 5′ end by substantiallyhomopolymeric nucleic acid sequences of 10 to 30 nucleotides in length.Such preferred target capture oligomers can be diagrammed as: 5′ X.sub.na′ b′ c′ Y.sub.n 3′, in which X.sub.n indicates substantiallyhomopolymeric sequence X that comprises n residues, a′ b′ c′ indicatesthe target hybridizing sequence, and Y.sub.n indicates substantiallyhomopolymeric sequence Y that comprises n residues. Preferably, n=about10 to about 30 contiguous nucleotides in length. The X.sub.n and Y.sub.nsequences are configured to form a double-stranded stem structure sothat the entire capture probe forms a hairpin structure with the targethybridizing sequence as the loop. Examples of hairpin capture probesinclude a 5′ poly-dT region adjacent to the target hybridizing regionand a 3′ poly-dA region so that the target hybridizing region forms theloop of the hairpin structure when the poly-dT and poly-dA regions arebound to each other, as illustrated in FIG. 1. In this embodiment, thetarget hybridizing region remains substantially single-stranded in thehairpin structure. Those skilled in the art will understand that anycomplementary sequences located in the 5′ and 3′ regions may be used toflank the target hybridizing region in an oligomer that forms a hairpincapture probe structure.

Other capture probe embodiments include partially or completelydouble-stranded structures made up of two oligomer strands in which atleast a portion of each of the individual single strands iscomplementary to a portion of the opposing single strand. Suchembodiments can be diagramed as:

-   -   (first strand) 5′ X.sub.n a′ b′ c′ 3′    -   (second strand) 3′ Y.sub.n a b c 5′        in which a′ b′ c′ indicates the target hybridizing sequence and        a b c indicates an optional sequence complementary to the target        hybridizing sequence, and in which the first and second strands        can form a double-stranded structure. Such a capture probe is        illustrated in FIG. 2. Other embodiments of capture probes are        single-stranded oligomers made up of a 3′ or 5′ target        hybridizing region and a contiguous region that binds to an        immobilized probe, which can be diagramed as: 5′ X.sub.n a′ b′        c′ 3′; or 5′ a′ b′ c′ X.sub.n 3′, in which X.sub.n indicates        sequence X that comprises n residues, and a′ b′ c′ indicates the        target hybridizing sequence.

A “sample” or “biological sample” refers to any composition or mixturein which a target nucleic acid of interest may be present, including butnot limited to plant or animal materials, waste materials, materials forforensic analysis, any tissue, cell, or extract derived from a living ordead organism which may contain a target nucleic acid, e.g., peripheralblood, bone marrow, plasma, serum, biopsy tissue including prostate,respiratory tissue or exudates, gastrointestinal tissue, urine, feces,semen, or other body fluids, and the like.

“Separating” or “isolating” or “purifying” refers to removing one ormore components from a complex mixture, such as a sample. Samplecomponents may include target and non-target nucleic acids, and othermaterials such as salts, acids, bases, detergents, proteins,carbohydrates, lipids and other organic or inorganic materials.Preferably, a separating, isolating or purifying step removes at least70%, preferably at least 90%, and more preferably about 95% of thetarget nucleic acids away from other sample components. A separating,isolating or purifying step may optionally include additional washingsteps to remove non-target sample components.

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating atarget nucleic acid from a target capture probe, and/or a target captureprobe from an immobilized probe. Release of the target nucleic acidstrand separates the target from other components of a capture hybridand makes the target available for binding to a detection probe oramplification oligomer. Other components of the capture hybrid mayremain bound, e.g., the capture probe strand to the immobilized probe ona capture support, without affecting target detection. Release of one ormore capture hybrid components may be accomplished by changing one ormore conditions to promote dissociation of components (e.g., heating toa certain Tm, changing salt concentrations, adding denaturants orcompetitive binding moieties to the mixture), or by using other wellknown methods such as strand displacement.

“Consisting essentially of” is used to mean that additionalcomponent(s), composition(s) or method step(s) that do not materiallychange the basic and novel characteristics of the present invention maybe included in the compositions, kits or methods of the presentinvention. Such characteristics include the ability of a targethybridizing sequence of an oligomer to bind or hybridize specifically toa target nucleic acid in a sample, the ability of a capture hybrid to beseparated from other sample components, or the ability of a detectionprobe to hybridize to the target nucleic acid and provide a detectablesignal to indicate the presence of the target in a sample.

The term “configured to” denotes an actual arrangement of a reagent. Forexample, when used to discuss an oligonucleotide, configured to denotesan actual arrangement of the polynucleotide sequence configuration ofthe referenced oligonucleotide. An oligonucleotide configured to form ahairpin structure means that the nucleotide sequence of theoligonucleotide is arranged to form a stem portion and a loop portionunder a set of conditions. Similarly, if two different oligonucleotidesare configured to hybridize to a target sequence under a first or asecond set of conditions it means that the nucleotide sequences of thetwo oligonucleotides are arranged to each hybridize to the targetsequences under its defined set of conditions. When the term is used todiscuss a set of conditions, then it denotes an actual arrangement ofchemical and/or physical properties. For example, a set of conditionsconfigured to cause a nucleic acid sequence to hybridize to anothernucleic acid sequence, can mean an actual range of temperatures underwhich the hybridizations occurs.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid (“target hybridizingsequence”), and which hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.An example of an amplification oligomer is a “primer” that hybridizes toa target nucleic acid and contains a 3′ OH end that is extended by apolymerase in an amplification process. Another example of anamplification oligomer is a “promoter-based amplification oligomer,”which comprises a target hybridizing sequence, and a promoter sequencefor initiating transcription by an appropriate polymerase.Promoter-based amplification oligomers may or may not be extendable by apolymerase in a primer-based extension depending upon whether or not the3′ end of the target hybridizing sequence is modified to preventprimer-based extension (e.g., a 3′ blocked end). A promoter-basedamplification oligonucleotide comprising a target hybridizing sequencethat is not modified to prevent primer-based extension is referred to asa “promoter-primer.” A promoter-based amplification oligonucleotidecomprising a target hybridizing sequence that is modified to preventprimer-based extension is referred to as a “promoter-provider.” Anotherexample of an amplification oligomer is an extender primer comprising a3′ target hybridizing sequence and a 5′ extension sequence. Preferably,but not necessarily, the extension sequence is configured to form a stemand a loop. Size ranges for amplification oligonucleotides include thosecomprising target hybridizing sequences that are about 8 to about 70 ntlong. Included in this range are all whole numbers of the range, as isunderstood by a skilled artisan (e.g., 8, 9, 10, 11, 12, 13 . . . 67,68, 69 and 70).

Oligomers not intended for primer-based extension by a nucleic acidpolymerase may include a blocker group that replaces the 3′OH to preventthe enzyme-mediated extension of the oligomer in an amplificationreaction. In some embodiments a blocking group near the 3′ end and maybe within five residues of the 3′ end and is sufficiently large to limitbinding of a polymerase to the oligomer. In other embodiments a blockinggroup is covalently attached to the 3′ terminus. Many different chemicalgroups may be used to block the 3′ end, e.g., alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter-provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter-provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The target-hybridizing portion of a promoteroligonucleotide is typically at least 10 nucleotides in length, and mayextend up to 50 or more nucleotides in length. The “second region”comprises a promoter sequence for an RNA polymerase. A promoter-provideroligonucleotide is configured so that it is incapable of being extendedby an RNA- or DNA-dependent DNA polymerase, (e.g., reversetranscriptase), preferably by comprising a blocking moiety at its3′-terminus as described above. This modification differentiatespromoter providers from promoter primers. Preferably, the promoterportion of a promoter primer or provider is a promoter for aDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6, though other promoters or modified version thereof can be used aswell.

By “preferentially hybridize” is meant that under stringenthybridization assay conditions, an oligonucleotide hybridizes to itstarget sequences, or replicates thereof, to form stable oligonucleotide:target sequence hybrid, while at the same time formation of stableoligonucleotide: non-target sequence hybrid is minimized. For example, aprobe oligonucleotide preferentially hybridizes to a target sequence orreplicate thereof to a sufficiently greater extent than to a non-targetsequence, to enable one having ordinary skill in the art to accuratelydetect the RNA replicates or complementary DNA (cDNA) of the targetsequence formed during the amplification. Appropriate hybridizationconditions are well known in the art for probe, amplification, targetcapture, blocker and other oligonucleotides, may be predicted based onsequence composition, or can be determined by using routine testingmethods (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual,2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly§§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

FIG. 1 illustrates a method embodiment that uses a capture probeconfigured to form a hairpin structure that is partially double-strandedand contains a single-stranded loop region that includes the targethybridizing sequence. The capture probe comprises complementary 5′ and3′ sequences (shown as poly-T and poly-A), which are configured to formthe double-stranded stem portion of the hairpin. These homopolymericsequences flank the target hybridizing sequence (shown as a′ b′ c′ d′,wherein a, b, c & d are merely indicators that a sequence is present,but alone do not denote and nucleotide arrangement, and wherein thepresence or absence of ′ indicates polarity such that a′ b′ c′ d′ is anucleic acid sequence that is substantially complementary to a b c d),which is configured to form the loop. In this embodiment, the 5′ and 3′ends of the hairpin capture probe are separated under a set ofconditions (e.g., heating above the Tm to dissociate the hydrogen bonds)to make a linear single-stranded capture probe. The now linear captureprobe hybridizes with a target nucleic acid (shown as sequence d c b a)under a set of conditions. If the target nucleic acid is adouble-stranded molecule, then it first can be dissociated into singlestranded molecules using standard chemical or physical methods. Forexample, the double strands of the target nucleic acid can be melted ina single melting step that linearizes the capture probe and dissociatesdouble stranded target nucleic acid strands before hybridization of thecapture probe and the target strand. Following capture of the targetnucleic acid by the capture probe, excess capture probe, e.g., those nothybridized to target nucleic acids, reform the hairpin structure under aset of conditions to effectively prevent non-specific binding of theseexcess capture probes to other components in the mixture, or to theimmobilized probe.

In this embodiment, the capture hybrid is made up of the target nucleicacid hybridized to the target hybridizing sequence of the capture probeand one of the substantially heteropolymeric sequences of the captureprobe (here illustrated as a 3′ poly-A) hybridized to a complementaryimmobilized probe (here illustrated as a poly-T) attached to the capturesupport. One or more of these hybridization steps can be performedsequentially or simultaneously depending on the conditions used and/orthe configuration of these oligonucleotides. Following formation of thecapture hybrid, the capture hybrid is isolated from other samplecomponents, including excess hairpin capture probes, non-target nucleicacids and cellular debris, by physically separating the capture supportfrom these components using any of a variety of known methods, e.g.,centrifugation, filtration, magnetic attraction of a magnetic capturesupport. To further facilitate isolation of the target nucleic acid fromother sample components that adhere non-specifically to any portion ofthe capture hybrid, the capture hybrid may be washed one or more timesto dilute and remove other sample components. Washing may beaccomplished by dissociating the capture hybrid into its individualcomponents in an appropriate aqueous solution (e.g., 10 mM Tris, 1 mMEDTA) and appropriate conditions (e.g., temperature above the Tm of thecomponents) and then readjusting the conditions to permit reformation ofthe capture hybrid. For ease of handling and minimization of steps,washing preferably rinses the intact capture hybrid attached to thecapture support in a solution by using conditions that maintain thecapture hybrid.

The method next proceeds by releasing the target from the capturehybrid. For example, the capture hybrid is released into its individualcomponents to free the target nucleic acid into solution, making itavailable to hybridize with a detection probe or a first primeroligomer, depending on whether or not an amplification step is to beperformed before detection. In FIG. 1 the capture probe and targetnucleic acid are illustrated as being released into a solution fordetection of the target nucleic acid. The capture probe and thedetection probe are illustrated as comprising the same targethybridizing sequence, and thus can compete for hybridization to thetarget nucleic acid. To prevent competition by the capture probe forhybridizing to the target nucleic acid, a set of conditions are providedfor forming a hairpin structured capture probe. The detection probe isthen hybridized to the target under a set of conditions and the presenceor absence or abundance of the target nucleic acid is determined. Anintervening amplification step could have followed the step releasingthe capture probe and target nucleic acid into solution. Anamplification step requires that a first amplification oligomerhybridize the target nucleic acid. Here, the amplification oligomer andthe capture probe both comprise target hybridizing sequences thatcompete for hybridizing the small target nucleic acid, thus theseoligonucleotides comprise substantially identical target hybridizingsequences, or at least target hybridizing sequences configured tohybridize to overlapping target sequences on the small target nucleicacid. To prevent competition by the capture probe for hybridizing to thetarget nucleic acid, a set of conditions are provided for forming ahairpin structured capture probe. The amplification oligomer is thenhybridized to the target nucleic acid under a set of conditions and thetarget nucleic acid is amplified. The second amplification oligomer maycomprise a target hybridizing sequence that is complementary to all or aportion of the target capture oligomer target hybridizing sequence.Thus, the target capture oligomer can interfere with the secondamplification oligomer hybridizing to its intended target sequence.

FIG. 2 illustrates a method embodiment in which the target nucleic acidis captured by using a completely or partially double-stranded captureprobe that contains complementary sequences on two strands (shown as a3′ poly-A region on one strand and a 5′ poly-T region on the otherstrand) and at least one target hybridizing sequence (shown as a′ b′ c′on the poly-T containing strand). In this embodiment, only one strand ofthe capture probe hybridizes to the target nucleic acid (shown assequence a b c d e). It is important that the capture probe strand thatbinds to the target sequence also contains a specific binding partnermember that binds to the immobilized probe (shown as a poly-A strand onthe capture support). The partially double-stranded capture probe isusually dissociated before forming the capture hybrid although stranddisplacement caused by the target binding to the target hybridizingsequence of the capture probe may separate the capture probe strands.FIG. 2 illustrates a completely double-stranded embodiment in which thetwo strands contain portions that are complementary to each other (shownas poly-A on one strand, and poly-T on the other strand), and one strandcontains a target hybridizing sequence whereas the other strand containa sequence complementary to the target hybridizing sequence. Thoseskilled in the art will appreciate that the capture probe may bepartially double-stranded (e.g., substituting a polyA strand for thepolyA-a b c strand shown in FIG. 2). For completely and partiallydouble-stranded capture probes, the same assay steps are used,optionally starting with separation of the capture probe strands usingstandard methods to allow hybridization of the target hybridizingportion of one capture probe strand to the target nucleic acid. Becausethe two capture probe strands can rehybridize (e.g., via poly-A bindingto polyT) and interfere with the target hybridizing sequence of thecapture probe strand binding to the target nucleic acid, those skilledin the art will appreciate that the capture probe may be synthesizedwith modifications to optimize hybridization to the target nucleic acid.

In this embodiment, the capture hybrid is made up of the target nucleicacid hybridized to the target hybridizing sequence of one capture probestrand, and another portion of the same capture probe strand (shown aspoly-T) is bound to an immobilized probe (shown as poly-A) attached to acapture support. As described above, the capture hybrid attached to thesupport is separated from other sample components and, optionally,washed to remove sample components and capture probe strands unbound tothe capture hybrid. Capture probe strands that do not bind to the targetsequence can reform the partially or completely double-strandedstructure and be washed away, along with unbound single strands. Then,the target nucleic acid is released from the capture hybrid or thecapture hybrid is separated into its components, and the released targetis bound in solution by a detection probe (shown as sequence a′ b′ c′ d′e′) to form a detection hybrid that produces a signal (shown as a starstructure) which is detected to indicate the presence of the targetnucleic acid in the sample. The released capture probe strand from thecapture hybrid remains in solution as illustrated in FIG. 2, but doesnot bind the detection probe because it is the same sense strand as thedetection probe. Similarly, if an amplification step is to be performedbefore the detection step, then the amplification oligomer comprises allor part of the a′b′c′d′e′ target hybridizing sequence and competes withthe capture probe for hybridizing the target nucleic acid. Those skilledin the art will appreciate that the detection probe or amplificationoligomer are configured to include structure that favors its binding tothe target nucleic acid (e.g., increased sequence length and/or backbonemodifications) to minimize competition between with the released captureprobe strand. In an embodiment, the released capture probe can be usedas a primer member in an amplification reaction. In one aspect, thereleased capture probe sequence can comprise a promoter sequence and canbe used as a promoter-based amplification oligomer.

A typical assay that uses a method described herein involves providing asample suspected of containing one or more small RNAs of interest. Sucha sample may be used directly in the assay or prepared by using any of avariety of methods, from simple dilution of a biological fluid with alysing solution to more complex methods that are well known in the art(e.g., Su et al., J. Mol. Diagn. 2004, 6:101-107; Sambrook, J. et al.,1989, Molecular Cloning, A Laboratory Manual, 2nd ed., pp. 7.37-7.57;and U.S. Pat. Nos. 5,374,522, 5,386,024, 5,786,208, 5,837,452, and6,551,778). Typically, a sample is heated to inactivate enzymes in thesample and to make the nucleic acids in the sample single-stranded(e.g., 90-100.deg.C. for 2-10 min, then rapidly cooling to 0-5.deg.C.).To form a capture hybrid, the sample is incubated in appropriatehybridization conditions in the presence of a capture probe (preferablyof a configuration described above) and an immobilized probe attached toa capture support. An efficient method mixes these components togetherin a hybridization reaction mixture and uses first conditions to promotehybridization between the target hybridizing sequence of a capture probeand the target nucleic acid, followed by second conditions to promotebinding of the capture probe:target nucleic acid complex to theimmobilized probe. For example, the first conditions may incubate thereaction mixture at a temperature below the Tm for the targethybridizing sequence of the capture probe and the target nucleic acidbut above the Tm for hybridization of sequences that bind the captureprobe and the immobilized probe, followed by incubating at a secondtemperature below the Tm for the capture probe binding to immobilizedprobe sequences (U.S. Pat. No. 6,110,678). In embodiments in which thecapture hybrid is attached to the capture support by using members of aspecific binding pair that do not require nucleic acid hybridization(e.g., biotin and avidin or streptavidin), appropriate conditions forthe selected binding pair members are used. An alternate approachincubates the target capture oligomer and the target nucleic acid underconditions configured to open the hairpin capture probe, hybridize thecapture probe and the target nucleic acid, and then reform a hairpinstructure for any unhybridized capture probe. Then, an immobilized probeand support are mixed into the reaction mixture in conditions configuredto hybridize the target capture oligomer:target nucleic acid complex andthe immobilized probe to obtain a capture hybrid. Following formation ofthe capture hybrids, the capture hybrids attached to the capture supportare separated physically from other sample components by using wellknown methods appropriate for the support, e.g., removing a filter,membrane, or particle from the solution phase by using filtration,centrifugation, gravity, magnetic force, and the like. When the capturesupport with attached capture hybrids have been separated from othersample components, optional washing steps may be included to furtherpurify the captured target nucleic acid, preferably performed whilemaintaining the capture hybrid attached to the capture support. Then thetarget nucleic acid or all components of the capture hybrid are releasedinto solution to free the target for the detection step. Release of thetarget or capture hybrid components may be performed by any knownmethod, such as, e.g., changing the temperature or chemical compositionof the mixture to promote dissociation of the capture hybrid into one ormore of its nucleic acid components. Typically, a simple heating step isperformed to melt the target and capture probe strands, e.g., in anaqueous solution of low ionic strength, at 90-100.deg.C. for 5 min,followed by rapid cooling to 0-5.deg.C. As illustrated in FIG. 1, thecapture probe is reformed via intramolecular hybridization to a hairpinform. The subsequent amplification and/or detection step may beperformed in soluble phase by adding a detection probe/amplificationoligomer directly to the soluble phase containing the released targetnucleic acid and incubating the mixture in hybridization conditionsconfigured for binding the detection probe/amplification oligomer andtarget sequences (e.g., adding salts to the soluble phase to make asolution of suitable ionic strength and incubating at 25-60.deg.C.).After the detection probe binds to the target nucleic acid to form thedetection hybrid, a signal from the hybrid is detected to indicate thepresence of the target in the tested sample.

The invention includes compositions useful for performing the methods ofdetecting target nucleic acids described herein. Some compositionsherein include one or more target capture oligomers for hybridizing to atarget nucleic acid. In some methods for detecting the presence orabundance of two or more target nucleic acids, composition includes twoor more target capture oligomers each configured to selectivelyhybridize each of their respective target nucleic acids. Some targetcapture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′format, wherein a′b′c′ is substantially complementary to all or aportion of a micro RNA and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some target capture oligomers are configured in a 5′ X.sub.n a′b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementaryto all or a portion of miR-21 and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some target capture oligomers are configured in a 5′ X.sub.n a′b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementaryto all or a portion of miR-34b and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some target capture oligomers are configured in a 5′ X.sub.n a′b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementaryto all or a portion of miR-182 and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some target capture oligomers are configured in a 5′ X.sub.n a′b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementaryto all or a portion of miR-221 and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some target capture oligomers are configured in a 5′ X.sub.n a′b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementaryto all or a portion of miR-222 and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some target capture oligomers are configured in a 5′ X.sub.n a′b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementaryto all or a portion of miR-802 and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother.

Some compositions herein include one or more first amplificationoligomers for hybridizing to a target nucleic acid. In some methods fordetecting the presence or abundance of two or more target nucleic acids,composition includes two or more first amplification oligomers eachconfigured to selectively hybridize each of their respective targetnucleic acids. Some first amplification oligomers are configured in a 5′Z.sub.n a′ b′ c′ 3′ format, wherein a′ b′ c′ is substantiallycomplementary to all or a portion of a micro RNA sequence and wherein Zis an extension nucleic acid sequence. The a′ b′ c′ region of the firstamplification oligomer can be substantially complementary to is theentire micro RNA sequence. The a′b′c′ region of the first amplificationoligomer can be substantially complementary to a portion of a micro RNA,wherein the portion is up to 20 contiguous nucleotides of the micro RNAsequence. The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 20 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 15 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 10 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 or 27 contiguous nucleotides of the micro RNAsequence. The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotides at the 3′ end ofthe micro RNA sequence. Preferably, the 5′ Z.sub.n region of the firstamplification primer is configured to form a hairpin structure. The 5′Z.sub.n region of the first amplifications primer can be configured tocontain a target sequence or complement thereof for hybridizing areverse primer oligomer or a detection probe oligomer or both. Somefirst amplification oligomers are configured in a 5′ Z.sub.n a′ b′ c′ 3′format, wherein a′b′c′ is substantially complementary to all or aportion of a miR-21 sequence and wherein Z is an extension nucleic acidsequence. Some first amplification oligomers are configured in a 5′Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of a miR-34b sequence and wherein Z isan extension nucleic acid sequence. Some first amplification oligomersare configured in a 5′ Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ issubstantially complementary to all or a portion of a miR-182 sequenceand wherein Z is an extension nucleic acid sequence. Some firstamplification oligomers are configured in a 5′ Z.sub.n a′ b′ c′ 3′format, wherein a′b′c′ is substantially complementary to all or aportion of a miR-221 sequence and wherein Z is an extension nucleic acidsequence. Some first amplification oligomers are configured in a 5′Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of a miR-222 sequence and wherein Z isan extension nucleic acid sequence. Some first amplification oligomersare configured in a 5′ Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ issubstantially complementary to all or a portion of a miR-802 sequenceand wherein Z is an extension nucleic acid sequence.

Some compositions herein include at least one promoter-based oligomerfor hybridizing to a cDNA containing the substantial complement of atarget nucleic acid sequence. In some methods for detecting the presenceor abundance of two or more target nucleic acids the compositionincludes two or more promoter-based oligomers each configured toselectively hybridize a cDNA containing the substantial complement theirrespective target nucleic acid. Some promoter-based oligomers areconfigured in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a micro RNA sequence andwherein the promoter is an RNA polymerase promoter sequence. The a b cregion of the promoter-based oligomer can be substantially identical tothe entire micro RNA sequence. The a b c region of the promoter-basedoligomer can be substantially identical to a portion of a micro RNA,wherein the portion is up to 20 contiguous nucleotides of the micro RNAsequence. The a b c region of the promoter-based oligomer can besubstantially identical to a portion of a micro RNA, wherein the portionis up to 20 contiguous nucleotides of the micro RNA sequence. The a b cregion of the promoter-based oligomer can be substantially identical toa portion of a micro RNA, wherein the portion is up to 15 contiguousnucleotides of the micro RNA sequence. The a b c region of thepromoter-based oligomer can be substantially identical to a portion of amicro RNA, wherein the portion is up to 10 contiguous nucleotides of themicro RNA sequence. The a b c region of the promoter-based oligomer canbe substantially identical to a portion of a micro RNA, wherein theportion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 or 27 contiguous nucleotides of the micro RNAsequence. The a b c region of the promoter-based oligomer can besubstantially identical to a portion of a micro RNA, wherein the portionis the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26 or 27 contiguous nucleotides at the 5′ end of themicro RNA sequence. Some promoter-based oligomers are configured in a 5′promoter a b c 3′ format, wherein a b c is substantially identical toall or a portion of a miR-21 sequence. Some promoter-based oligomers areconfigured in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-34b sequence. Somepromoter-based oligomers are configured in a 5′ promoter a b c 3′format, wherein a b c is substantially identical to all or a portion ofa miR-182 sequence. Some promoter-based oligomers are configured in a 5′promoter a b c 3′ format, wherein a b c is substantially identical toall or a portion of a miR-221 sequence. Some promoter-based oligomersare configured in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-222 sequence. Somepromoter-based oligomers are configured in a 5′ promoter a b c 3′format, wherein a b c is substantially identical to all or a portion ofa miR-802 sequence. Some promoter-based oligomers can optionallycomprise a tag sequence between the 5′ promoter region and the 3′ a b cregion. In some methods for amplification using a universalamplification oligomer, a first promoter-based oligomer comprises a tagsequence between the 5′ promoter region and the 3′ a b c region and asecond promoter-based oligomer is configured to hybridize to thecomplement of that tag sequence. In some methods for detecting thepresence or abundance of two or more target nucleic acids, thecomposition includes two or more promoter-based oligomers eachconfigured to selectively hybridize a cDNA containing the substantialcomplement their respective target nucleic acids, wherein each of thetwo or more promoter based oligomers comprise a tag sequence betweentheir 5′ promoter regions and their 3′ a b c regions. In some methodsfor detecting the presence or abundance of two or more target nucleicacids, the composition includes two or more promoter-based oligomerseach configured to selectively hybridize a cDNA containing thesubstantial complement their respective target nucleic acids, whereineach of the two or more promoter based oligomers comprise a tag sequencebetween their 5′ promoter regions and their 3′ a b c regions and whereineach of the tag sequences in each of these two or more promoter-basedoligomers are substantially identical. In some methods for detecting thepresence or abundance of two or more target nucleic acids, thecomposition includes two or more promoter-based oligomers eachconfigured to selectively hybridize a cDNA containing the substantialcomplement their respective target nucleic acids, wherein each of thetwo or more promoter based oligomers comprise a tag sequence betweentheir 5′ promoter regions and their 3′ a b c regions and wherein each ofthe tag sequences in each of these two or more promoter-based oligomersare substantially identical and wherein the composition further includesan additional promoter based oligomer comprising a target hybridizingsequence that is also substantially identical to the tag sequences. Somecompositions include at least one promoter-based oligomer that is apromoter primer. Some compositions include at least one promoter-basedoligomer that is a promoter provider.

Some compositions include at least one detection probe oligomer forhybridizing to a target nucleic acid sequence. In some methods fordetecting the presence or abundance of two or more target nucleic acids,the composition includes contain two or more detection probe oligomerseach configured to selectively hybridize each of their respective targetnucleic acids. Some detection probe oligomers for hybridizing to atarget nucleic acid sequence are linear probes. Some detection probeoligomers are labeled linear probes. Some detection probe oligomers aredual labeled linear probes. Some detection probe oligomers are hairpinprobes. Some detection probe oligomers are labeled hairpin probe. Somedetection probe oligomers are dual labeled hairpin probe. Some detectionprobe oligomers are molecular beacons. Some detection probe oligomersare molecular torches. Some detection probe oligomers comprise a targethybridizing region that is substantially complementary to all or aportion of a micro RNA. Some detection probe oligomers comprise a targethybridizing region that is substantially complementary to a portion of amicro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotidesof the micro RNA. Some detection probe oligomers comprise a targethybridizing region that is substantially complementary to a portion of amicro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotidesat the 3′ end of the micro RNA. Some detection probe oligomers comprisea target hybridizing region that is substantially complementary to aportion of a micro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27contiguous nucleotides at the 5′ end of the micro RNA. Some detectionprobe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-21. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-34b. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-182. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-221. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-222. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-802. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template using anamplification oligomer described herein. Some detection probe oligomersare configured to hybridize to an amplification product generated from amicro RNA template using an amplification oligomer selected from a firstamplification oligomer as described herein, a promoter-based oligomer asdescribed herein, a reverse primer oligomer as described herein orcombinations thereof. Some detection probe oligomers are configured tohybridize to an amplification product generated from a micro RNAtemplate using a first amplification oligomer in a 5′ Z.sub.n a′ b′ c′3′ format as described herein, wherein the detection probe oligomercomprises a target hybridizing sequence that is substantiallycomplementary to a portion of the extension nucleic acid sequence. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template using a firstamplification oligomer in a 5′ Z.sub.n a′ b′ c′ 3′ format as describedherein, wherein the detection probe oligomer comprises a targethybridizing region that is substantially identical to all or a portionof the micro RNA template. Some detection probe oligomers are configuredto hybridize to an amplification product generated from a micro RNAtemplate using a first amplification oligomer in a 5′ Z.sub.n a′ b′ c′3′ format as described herein, wherein the detection probe oligomercomprises a target hybridizing region that is substantiallycomplementary to a portion of the extension nucleic acid sequence and toall or a portion of the amplification product sequence that is itselfcomplementary to the micro RNA template. Some detection probe oligomersare configured to hybridize to an amplification product generated from amicro RNA template using a promoter-based oligomer as described herein,wherein the detection probe oligomer comprises a target hybridizingsequence that is substantially identical to all or a portion of thetarget hybridizing sequence of the promoter-based oligomer. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template using apromoter-based oligomer containing a tag sequence as described herein,wherein the detection probe oligomer comprises a target hybridizingsequence that is substantially identical to all or a portion of thetarget hybridizing sequence of the promoter-based oligomer, all or aportion of the tag sequence or a combination thereof. Some detectionprobe oligomers are configured to hybridize to an amplification productgenerated from a micro RNA template using a promoter-based oligomer asdescribed herein, wherein the detection probe oligomer comprises atarget hybridizing region that is substantially complementary to an RNAtranscript amplification product.

The invention also includes kits containing components for performingthe methods for detecting target nucleic acids described herein. Somekits contain at least one target capture oligomer for hybridizing to atarget nucleic acid. Some kits for detecting the presence or abundanceof two or more target nucleic acids contain two or more target captureoligomers each configured to selectively hybridize each of theirrespective target nucleic acids. Some kits contain at least one targetcapture oligomer in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′ format, whereina′b′c′ is substantially complementary to all or a portion of a micro RNAand wherein X and Y are substantially homopolymeric nucleic acidsequences that are complementary one to the other. Some kits contain atlease one target capture oligomer in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′format, wherein a′b′c′ is substantially complementary to all or aportion of miR-21 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Somekits contain at lease one target capture oligomer in a 5′ X.sub.n a′ b′c′ Y.sub.n 3′ format, wherein a′b′c′ is substantially complementary toall or a portion of miR-34b and wherein X and Y are substantiallyhomopolymeric nucleic acid sequences that are complementary one to theother. Some kits contain at lease one target capture oligomer in a 5′X.sub.n a′ b′ c′ Y.sub.n 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of miR-182 and wherein X and Y aresubstantially homopolymeric nucleic acid sequences that arecomplementary one to the other. Some kits contain at lease one targetcapture oligomer in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′ format, whereina′b′c′ is substantially complementary to all or a portion of miR-221 andwherein X and Y are substantially homopolymeric nucleic acid sequencesthat are complementary one to the other. Some kits contain at lease onetarget capture oligomer in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′ format,wherein a′b′c′ is substantially complementary to all or a portion ofmiR-222 and wherein X and Y are substantially homopolymeric nucleic acidsequences that are complementary one to the other. Some kits contain atlease one target capture oligomer in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′format, wherein a′b′c′ is substantially complementary to all or aportion of miR-802 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other.

Some kits contain at least one first amplification oligomer forhybridizing to a target nucleic acid. Some kits for detecting thepresence or abundance of two or more target nucleic acids contain two ormore first amplification oligomers each configured to selectivelyhybridize each of their respective target nucleic acids. Some kitscontain at least one first amplification oligomer in a 5′ Z.sub.n a′ b′c′ 3′ format, wherein a′ b′ c′ is substantially complementary to all ora portion of a micro RNA sequence and wherein Z is an extension nucleicacid sequence. Preferably, the a′ b′ c′ region of the firstamplification oligomer is substantially complementary to is the entiremicro RNA sequence. Preferably, the a′b′c′ region of the firstamplification oligomer is substantially complementary to a portion of amicro RNA, wherein the portion is up to 20 contiguous nucleotides of themicro RNA sequence. Preferably, the a′b′c′ region of the firstamplification oligomer is substantially complementary to a portion of amicro RNA, wherein the portion is up to 20 contiguous nucleotides of themicro RNA sequence. Preferably, the a′b′c′ region of the firstamplification oligomer is substantially complementary to a portion of amicro RNA, wherein the portion is up to 15 contiguous nucleotides of themicro RNA sequence. Preferably, the a′b′c′ region of the firstamplification oligomer is substantially complementary to a portion of amicro RNA, wherein the portion is up to 10 contiguous nucleotides of themicro RNA sequence. Preferably, the a′b′c′ region of the firstamplification oligomer is substantially complementary to a portion of amicro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotidesof the micro RNA sequence. Preferably, the a′b′c′ region of the firstamplification oligomer is substantially complementary to a portion of amicro RNA, wherein the portion is the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguousnucleotides at the 3.' end of the micro RNA sequence. Preferably, the5′Z.sub.n region of the first amplification primer is configured to forma hairpin structure. Preferably, the 5′Z.sub.n region of the firstamplifications primer is configured to contain a target sequence orcomplement thereof for hybridizing a reverse primer oligomer or adetection probe oligomer or both. Some kits contain at least one firstamplification oligomer in a 5′ Z.sub.n a′ b′ c′ 3′ format, whereina′b′c′ is substantially complementary to all or a portion of a miR-21sequence and wherein Z is an extension nucleic acid sequence. Some kitscontain at least one first amplification oligomer in a 5′ Z.sub.n a′ b′c′ 3′ format, wherein a′b′ c′ is substantially complementary to all or aportion of a miR-34b sequence and wherein Z is an extension nucleic acidsequence. Some kits contain at least one first amplification oligomer ina 5′ Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of a miR-182 sequence and wherein Z isan extension nucleic acid sequence. Some kits contain at least one firstamplification oligomer in a 5′ Z.sub.n a′ b′ c′ 3′ format, whereina′b′c′ is substantially complementary to all or a portion of a miR-221sequence and wherein Z is an extension nucleic acid sequence. Some kitscontain at least one first amplification oligomer in a 5′ Z.sub.n a′ b′c′ 3′ format, wherein a′b′c′ is substantially complementary to all or aportion of a miR-222 sequence and wherein Z is an extension nucleic acidsequence. Some kits contain at least one first amplification oligomer ina 5′ Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of a miR-802 sequence and wherein Z isan extension nucleic acid sequence.

Some kits contain at least one promoter-based oligomer for hybridizingto a cDNA containing the substantial complement of a target nucleic acidsequence. Some kits for detecting the presence or abundance of two ormore target nucleic acids contain two or more promoter-based oligomerseach configured to selectively hybridize a cDNA containing thesubstantial complement their respective target nucleic acid. Some kitscontain at least one promoter-based oligomer in a 5′ promoter a b c 3′format, wherein a b c is substantially identical to all or a portion ofa micro RNA sequence and wherein the promoter is an RNA polymerasepromoter sequence. Preferably, the a b c region of the promoter-basedoligomer is substantially identical to the entire micro RNA sequence.Preferably, the a b c region of the promoter-based oligomer issubstantially identical to a portion of a micro RNA, wherein the portionis up to 20 contiguous nucleotides of the micro RNA sequence.Preferably, the a b c region of the promoter-based oligomer issubstantially identical to a portion of a micro RNA, wherein the portionis up to 20 contiguous nucleotides of the micro RNA sequence.Preferably, the a b c region of the promoter-based oligomer issubstantially identical to a portion of a micro RNA, wherein the portionis up to 15 contiguous nucleotides of the micro RNA sequence.Preferably, the a b c region of the promoter-based oligomer issubstantially identical to a portion of a micro RNA, wherein the portionis up to 10 contiguous nucleotides of the micro RNA sequence.Preferably, the a b c region of the promoter-based oligomer issubstantially identical to a portion of a micro RNA, wherein the portionis 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26 or 27 contiguous nucleotides of the micro RNA sequence.Preferably, the a b c region of the promoter-based oligomer issubstantially identical to a portion of a micro RNA, wherein the portionis the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26 or 27 contiguous nucleotides at the 5′ end of themicro RNA sequence. Some kits contain at least one promoter-basedoligomer in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-21 sequence. Somekits contain at least one promoter-based oligomer in a 5′ promoter a b c3′ format, wherein a b c is substantially identical to all or a portionof a miR-34b sequence. Some kits contain at least one promoter-basedoligomer in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-182 sequence. Somekits contain at least one promoter-based oligomer in a 5′ promoter a b c3′ format, wherein a b c is substantially identical to all or a portionof a miR-221 sequence. Some kits contain at least one promoter-basedoligomer in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-222 sequence. Somekits contain at least one promoter-based oligomer in a 5′ promoter a b c3′ format, wherein a b c is substantially identical to all or a portionof a miR-802 sequence. Some kits can optionally contain a tag sequencebetween the 5′ promoter region and the 3′ a b c region. Some kits canoptionally contain a tag sequence between the 5′ promoter region and the3′ a b c region and a secondary promoter-based oligomer configured tohybridize to the complement of the tag sequence. Some kits for detectingthe presence or abundance of two or more target nucleic acids containtwo or more promoter-based oligomers each configured to selectivelyhybridize a cDNA containing the substantial complement their respectivetarget nucleic acids, wherein each of the two or more promoter basedoligomers contain a tag sequence between their 5′ promoter regions andtheir 3′ a b c regions. Some kits for detecting the presence orabundance of two or more target nucleic acids contain two or morepromoter-based oligomers each configured to selectively hybridize a cDNAcontaining the substantial complement their respective target nucleicacids, wherein each of the two or more promoter based oligomers containa tag sequence between their 5′ promoter regions and their 3′ a b cregions and wherein each of the tag sequences in each of these two ormore promoter-based oligomers are substantially identical. Some kits fordetecting the presence or abundance of two or more target nucleic acidscontain two or more promoter-based oligomers each configured toselectively hybridize a cDNA containing the substantial complement theirrespective target nucleic acids, wherein each of the two or morepromoter based oligomers contain a tag sequence between their 5′promoter regions and their 3′ a b c regions and wherein each of the tagsequences in each of these two or more promoter-based oligomers aresubstantially identical and wherein the kit further contains anadditional promoter based oligomer comprising a target hybridizingsequence that is also substantially identical to the tag sequence. Somekits contain at least one promoter-based oligomer that is a promoterprimer. Some kits contain at least one promoter-based oligomer that is apromoter provider.

Some kits contain at least one detection probe oligomer for hybridizingto a target nucleic acid sequence. Some kits for detecting the presenceor abundance of two or more target nucleic acids contain two or moredetection probe oligomers each configured to selectively hybridize eachof their respective target nucleic acids. Some kits contain at least onedetection probe oligomer for hybridizing to a target nucleic acidsequence, wherein the detection probe oligomer is a linear probe. Somekits contain at least one detection probe oligomer for hybridizing to atarget nucleic acid sequence, wherein the detection probe oligomer is alabeled linear probe. Some kits contain at least one detection probeoligomer for hybridizing to a target nucleic acid sequence, wherein thedetection probe oligomer is a dual labeled linear probe. Some kitscontain at least one detection probe oligomer for hybridizing to atarget nucleic acid sequence, wherein the detection probe oligomer is ahairpin probe. Some kits contain at least one detection probe oligomerfor hybridizing to a target nucleic acid sequence, wherein the detectionprobe oligomer is a labeled hairpin probe. Some kits contain at leastone detection probe oligomer for hybridizing to a target nucleic acidsequence, wherein the detection probe oligomer is a dual labeled hairpinprobe. Some kits contain at least one detection probe oligomer forhybridizing to a target nucleic acid sequence, wherein the detectionprobe oligomer is a molecular beacon. Some kits contain at least onedetection probe oligomer for hybridizing to a target nucleic acidsequence, wherein the detection probe oligomer is a molecular torch.Some kits contain at least one detection probe oligomer comprising atarget hybridizing region that is substantially complementary to all ora portion of a micro RNA. Some kits contain at least one detection probeoligomer comprising a target hybridizing region that is substantiallycomplementary to a portion of a micro RNA, wherein the portion is 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26 or 27 contiguous nucleotides of the micro RNA. Some kits contain atleast one detection probe oligomer comprising a target hybridizingregion that is substantially complementary to a portion of a micro RNA,wherein the portion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotides at the3′ end of the micro RNA. Some kits contain at least one detection probeoligomer comprising a target hybridizing region that is substantiallycomplementary to a portion of a micro RNA, wherein the portion is 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26 or 27 contiguous nucleotides at the 5′ end of the micro RNA. Somekits contain at least one detection probe oligomer comprising a targethybridizing region that is substantially complementary to all or aportion of miR-21. Some kits contain at least one detection probeoligomer comprising a target hybridizing region that is substantiallycomplementary to all or a portion of miR-34b. Some kits contain at leastone detection probe oligomer comprising a target hybridizing region thatis substantially complementary to all or a portion of miR-182. Some kitscontain at least one detection probe oligomer comprising a targethybridizing region that is substantially complementary to all or aportion of miR-221. Some kits contain at least one detection probeoligomer comprising a target hybridizing region that is substantiallycomplementary to all or a portion of miR-222. Some kits contain at leastone detection probe oligomer comprising a target hybridizing region thatis substantially complementary to all or a portion of miR-802. Some kitscontain at least one detection probe oligomer for hybridizing to anamplification product generated from a micro RNA template. Some kitscontain at least one detection probe oligomer for hybridizing to anamplification product generated from a micro RNA template using anamplification oligomer described herein. Some kits contain at least onedetection probe oligomer for hybridizing to an amplification productgenerated from a micro RNA template using an amplification oligomerselected from a first amplification oligomer as described herein, apromoter-based oligomer as described herein, a reverse primer oligomeras described herein or combinations thereof. Some kits contain at leastone detection probe oligomer for hybridizing to an amplification productgenerated from a micro RNA template using a first amplification oligomerin a 5′ Z.sub.n a′ b′ c′ 3′ format as described herein, wherein thedetection probe oligomer comprises a target hybridizing sequence that issubstantially complementary to a portion of the extension nucleic acidsequence. Some kits contain at least one detection probe oligomer forhybridizing to an amplification product generated from a micro RNAtemplate using a first amplification oligomer in a 5′ Z.sub.n a′ b′ c′3′ format as described herein, wherein the detection probe oligomercomprises a target hybridizing region that is substantially identical toall or a portion of the micro RNA template. Some kits contain at leastone detection probe oligomer for hybridizing to an amplification productgenerated from a micro RNA template using a first amplification oligomerin a 5′ Z.sub.n a′ b′ c′ 3′ format as described herein, wherein thedetection probe oligomer comprises a target hybridizing region that issubstantially complementary to a portion of the extension nucleic acidsequence and to all or a portion of the amplification product sequencethat is itself complementary to the micro RNA template. Some kitscontain at least one detection probe oligomer for hybridizing to anamplification product generated from a micro RNA template using apromoter-based oligomer as described herein, wherein the detection probeoligomer comprises a target hybridizing sequence that is substantiallyidentical to all or a portion of the target hybridizing sequence of thepromoter-based oligomer. Some kits contain at least one detection probeoligomer for hybridizing to an amplification product generated from amicro RNA template using a promoter-based oligomer containing a tagsequence as described herein, wherein the detection probe oligomercomprises a target hybridizing sequence that is substantially identicalto all or a portion of the target hybridizing sequence of thepromoter-based oligomer, all or a portion of the tag sequence or acombination thereof. Some kits contain at least one detection probeoligomer for hybridizing to an amplification product generated from amicro RNA template using a promoter-based oligomer as described herein,wherein the detection probe oligomer comprises a target hybridizingregion that is substantially complementary to an RNA transcriptamplification product.

Some kits contain chemical compounds used in performing the methodsherein, such as enzymes, substrates, acids or bases to adjust pH of amixture, salts, buffers, chelating agents, denaturants, samplepreparation agents, sample storage or transport medium, cellular lysingagents, total RNA isolation components and reagents, partial generalizedRNA isolation components and reagents, solid supports, and otherinorganic or organic compounds. Kits may include any combination of theherein mentioned components and other components not mentioned herein.Components of the kits can be packaged in combination with each other,either as a mixture or in individual containers. It will be clear toskilled artisans that the invention includes many different kitconfigurations.

The invention includes methods for detecting the presence or abundanceof small RNA target nucleic acids described herein. The methods providehighly sensitive and selective detection of small RNA target nucleicacids from a sample. The methods provide highly sensitive and highlyselective detection of biomarkers for diagnosing a condition, making aprognosis about a condition, monitoring a therapy for a condition ordetermining a treatment for a condition. The methods provide highlysensitive and highly selective detection of micro RNA target nucleicacids for diagnosing a condition, making a prognosis about a condition,monitoring a therapy for a condition or determining a treatment for aconditions. The methods provide highly sensitive and highly selectivedetection of miR-21, miR-34b, miR-182, miR-221, miR-222, miR802 orcombinations thereof for diagnosing a condition, making a prognosisabout a condition, monitoring a therapy for a condition or determining atreatment for a conditions. The methods provide highly sensitive andhighly selective detection of biomarkers in a sample suspected toindicate a cancer, for diagnosing a cancer, making a prognosis about acancer, monitoring a cancer therapy or determining a cancer treatment.The methods provide highly sensitive and highly selective detection ofbiomarkers in a sample useful for identifying prostate cancer, fordiagnosing prostate cancer, making a prognosis about prostate cancercancer, monitoring prostate cancer therapy or determining a prostatecancer treatment. The methods provide highly sensitive and highlyselective detection of biomarkers present in a sample for discriminatingnormal tissue and/or indolent tumors from aggressive or metastaticdisease. The methods provide highly sensitive and highly selectivedetection of biomarkers present in a sample for discriminating normalprostate tissue and/or indolent prostate cancer tumors from aggressiveor metastatic prostate cancer. The methods provide highly sensitive andhighly selective detection of miR-21, miR-221, miR-222 or combinationsthereof present in a sample for discriminating normal prostate tissueand/or indolent prostate cancer tumors from aggressive or metastaticprostate cancer. The methods provide highly sensitive and highlyselective detection of biomarkers from a plurality of different sampletypes for identifying sample types that are useful for diagnosing acondition, making a prognosis about a condition, monitoring a therapyfor a condition or determining a treatment for a condition. The methodsprovide highly sensitive and highly selective detection of micro RNAtarget nucleic acids from a plurality of different sample types foridentifying sample types that are useful for diagnosing a condition,making a prognosis about a condition, monitoring a therapy for acondition or determining a treatment for a conditions. The methodsprovide highly sensitive and highly selective detection of miR-21,miR-34b, miR-182, miR-221, miR-222, miR802 or combinations thereof froma plurality of different sample types for identifying sample types thatare useful for diagnosing a condition, making a prognosis about acondition, monitoring a therapy for a condition or determining atreatment for a conditions. The methods provide highly sensitive andhighly selective detection of biomarkers from a plurality of differentsample types for identifying the presence, abundance and/or abundance ofone or more biomarkers in a sample type, thereby generating a profilefor the sample type that can be used for diagnosing a condition, makinga prognosis about a condition, monitoring a therapy for a condition ordetermining a treatment for a condition. Methods herein can be uniplexreactions. Methods herein can be multiplex reactions. A plurality ofuniplex reactions can be run in parallel. Methods herein can includeinternal controls for reaction quality and/or for quantitation. Methodsherein can be uniplex reactions for the detection of one of miR-21,miR-34b, miR-182, miR-221, miR-222 or miR-802, and may optionallyinclude an internal control. Methods herein can be multiplex reactionsfor the detection of one or more of miR-21, miR-34b, miR-182, miR-221,miR-222, miR-802, and may optionally include an internal control.

Some methods herein include one or more target capture oligomers forhybridizing to a target nucleic acid. Some methods for detecting thepresence or abundance of two or more target nucleic acids can use two ormore target capture oligomers each configured to selectively hybridizeeach of their respective target nucleic acids. Some target captureoligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′ format,wherein a′b′c′ is substantially complementary to all or a portion of amicro RNA and wherein X and Y are substantially homopolymeric nucleicacid sequences that are complementary one to the other. Some targetcapture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n 3′format, wherein a′b′c′ is substantially complementary to all or aportion of miR-21 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Sometarget capture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n3′ format, wherein a′b′c′ is substantially complementary to all or aportion of miR-34b and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Sometarget capture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n3′ format, wherein a′b′c′ is substantially complementary to all ‘or aportion of miR-182 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Sometarget capture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n3′ format, wherein a′b′c′ is substantially complementary to all or aportion of miR-221 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Sometarget capture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n3′ format, wherein a′b′c′ is substantially complementary to all or aportion of miR-222 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Sometarget capture oligomers are configured in a 5′ X.sub.n a′ b′ c′ Y.sub.n3′ format, wherein a′b′c′ is substantially complementary to all or aportion of miR-802 and wherein X and Y are substantially homopolymericnucleic acid sequences that are complementary one to the other. Somemethods herein include capture of a target nucleic acid using a hairpintarget capture probe as described herein to isolate the target nucleicaway from cellular debris, total RNA or size fractionated population ofgeneral RNA. The isolated target nucleic acid is optionally subjected toa wash step. The isolated target nucleic acid is released from thetarget capture probe and an amplification reaction is performed using atleast one amplification oligomer, wherein the target capture oligomerand an amplification oligomer comprise target hybridizing sequences thatcompete for binding a target sequence of the target nucleic acid.Preferably, a set of conditions are applied to the reaction mixture toform the target capture oligomer to a hairpin configuration and then theamplification reaction is performed. Some methods herein includegenerating an amplification reaction mixture that comprises at least onetarget nucleic acid, at least one amplification oligomer and at leastone target capture probe in a hairpin configuration.

Some methods herein include one or more first amplification oligomersfor hybridizing to a target nucleic acid. Some methods for detecting thepresence or abundance of two or more target nucleic acids can use two ormore first amplification oligomers each configured to selectivelyhybridize each of their respective target nucleic acids. Some firstamplification oligomers are configured in a 5′ Z.sub.n a′ b′ c′ 3′format, wherein a′ b′ c′ is substantially complementary to all or aportion of a micro RNA sequence and wherein Z is an extension nucleicacid sequence. The a′ b′ c′ region of the first amplification oligomercan be substantially complementary to is the entire micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 20 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 20 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 15 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is up to 10 contiguous nucleotides of the micro RNA sequence.The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26 or 27 contiguous nucleotides of the micro RNAsequence. The a′b′c′ region of the first amplification oligomer can besubstantially complementary to a portion of a micro RNA, wherein theportion is the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotides at the 3′ end ofthe micro RNA sequence. Preferably, the 5′Z.sub.n region of the firstamplification primer is configured to form a hairpin structure. The5′Z.sub.n region of the first amplifications primer can be configured tocontain a target sequence or complement thereof for hybridizing areverse primer oligomer or a detection probe oligomer or both. Somefirst amplification oligomers are configured in a 5′ Z.sub.n a′ b′ c′ 3′format, wherein a′b′c′ is substantially complementary to all or aportion of a miR-21 sequence and wherein Z is an extension nucleic acidsequence. Some first amplification oligomers are configured in a 5′Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of a miR-34b sequence and wherein Z isan extension nucleic acid sequence. Some first amplification oligomersare configured in a 5′ Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ issubstantially complementary to all or a portion of a miR-182 sequenceand wherein Z is an extension nucleic acid sequence. Some firstamplification oligomers are configured in a 5′ Z.sub.n a′ b′ c′ 3′format, wherein a′b′c′ is substantially complementary to all or aportion of a miR-221 sequence and wherein Z is an extension nucleic acidsequence. Some first amplification oligomers are configured in a 5′Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ is substantiallycomplementary to all or a portion of a miR-222 sequence and wherein Z isan extension nucleic acid sequence. Some first amplification oligomersare configured in a 5′ Z.sub.n a′ b′ c′ 3′ format, wherein a′b′c′ issubstantially complementary to all or a portion of a miR-802 sequenceand wherein Z is an extension nucleic acid sequence. Some methods hereininclude generating a cDNA strand from a target nucleic acid that is asmall RNA, preferably a micro RNA, more preferably one of miR-21,miR-34b, miR-182, miR-221, miR-222 or miR-802. Some methods hereininclude generating a cDNA strand from a target nucleic acid that is asmall RNA, preferably a micro RNA, more preferably one of miR-21,miR-34b, miR-182, miR-221, miR-222 or miR-802, wherein the cDNA strandcomprises a complement of an extension nucleic acid sequence from anextender primer as described herein. Some methods include generating areaction mixture that comprises at least one first amplificationoligomer and at least one promoter based oligomer, wherein the firstamplification oligomer and the promoter-based oligomer each comprisetarget hybridizing sequence that target overlapping sequences on a smallRNA target nucleic acid and complement thereof, preferably a micro RNAtarget nucleic acid, more preferably one of miR-21, miR-34b, miR-182,miR-221, miR222 or miR-802. Some methods include generating a reactionmixture that comprises at least one first amplification oligomer and atleast one promoter based oligomer, wherein the first amplificationoligomer and the promoter-based oligomer each comprise targethybridizing sequence that target non-overlapping sequences on a smallRNA target nucleic acid and complement thereof, preferably a micro RNAtarget nucleic acid, more preferably one of miR-21, miR-34b, miR-182,miR-221, miR222 or miR-802. Some methods include performing anamplification reaction in a reaction mixture that comprises a targetcapture oligomer, a first amplification oligomer, a promoter-basedoligomer and a target nucleic acid wherein the first amplificationoligomer and the target capture oligomer comprise target hybridizingsequences that compete for binding a target sequence of the targetnucleic acid, and wherein the target capture oligomer and thepromoter-based oligomer comprise target hybridizing sequences that areat least partially substantially complementary one to the other. Somemethods include performing an amplification reaction in a reactionmixture that comprises a target capture oligomer, a first amplificationoligomer, a promoter-based oligomer and a target nucleic acid whereinthe first amplification oligomer and the target capture oligomercomprise target hybridizing sequences that compete for binding a targetsequence of the target nucleic acid, and wherein the target captureoligomer and the promoter-based oligomer comprise target hybridizingsequences that are at least partially substantially complementary one tothe other, and wherein the target nucleic acid in a hairpin form.

Some methods herein include at least one promoter-based oligomer forhybridizing to a cDNA containing the substantial complement of a targetnucleic acid sequence. Some methods for detecting the presence orabundance of two or more target nucleic acids can use two or morepromoter-based oligomers each configured to selectively hybridize a cDNAcontaining the substantial complement their respective target nucleicacid. Some promoter-based oligomers are configured in a 5′ promoter a bc 3′ format, wherein a b c is substantially identical to all or aportion of a micro RNA sequence and wherein the promoter is an RNApolymerase promoter sequence. The a b c region of the promoter-basedoligomer can be substantially identical to the entire micro RNAsequence. The a b c region of the promoter-based oligomer can besubstantially identical to a portion of a micro RNA, wherein the portionis up to 20 contiguous nucleotides of the micro RNA sequence. The a b cregion of the promoter-based oligomer can be substantially identical toa portion of a micro RNA, wherein the portion is up to 20 contiguousnucleotides of the micro RNA sequence. The a b c region of thepromoter-based oligomer can be substantially identical to a portion of amicro RNA, wherein the portion is up to 15 contiguous nucleotides of themicro RNA sequence. The a b c region of the promoter-based oligomer canbe substantially identical to a portion of a micro RNA, wherein theportion is up to 10 contiguous nucleotides of the micro RNA sequence.The a b c region of the promoter-based oligomer can be substantiallyidentical to a portion of a micro RNA, wherein the portion is 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26or 27 contiguous nucleotides of the micro RNA sequence. The a b c regionof the promoter-based oligomer can be substantially identical to aportion of a micro RNA, wherein the portion is the 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27contiguous nucleotides at the 5′ end of the micro RNA sequence. Somepromoter-based oligomers are configured in a 5′ promoter a b c 3′format, wherein a b c is substantially identical to all or a portion ofa miR-21 sequence. Some promoter-based oligomers are configured in a 5′promoter a b c 3′ format, wherein a b c is substantially identical toall or a portion of a miR-34b sequence. Some promoter-based oligomersare configured in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-182 sequence. Somepromoter-based oligomers are configured in a 5′ promoter a b c 3′format, wherein a b c is substantially identical to all or a portion ofa miR-221 sequence. Some promoter-based oligomers are configured in a 5′promoter a b c 3′ format, wherein a b c is substantially identical toall or a portion of a miR-222 sequence. Some promoter-based oligomersare configured in a 5′ promoter a b c 3′ format, wherein a b c issubstantially identical to all or a portion of a miR-802 sequence. Somepromoter-based oligomers can optionally comprise a tag sequence betweenthe 5′ promoter region and the 3′ a b c region. In some methods foramplification using a universal amplification oligomer, a firstpromoter-based oligomer comprises a tag sequence between the 5′ promoterregion and the 3′ a b c region and a second promoter-based oligomer isconfigured to hybridize to the complement of that tag sequence. In somemethods for detecting the presence or abundance of two or more targetnucleic acids, the composition includes two or more promoter-basedoligomers each configured to selectively hybridize a cDNA containing thesubstantial complement their respective target nucleic acids, whereineach of the two or more promoter based oligomers comprise a tag sequencebetween their 5′ promoter regions and their 3′ a b c regions. In somemethods for detecting the presence or abundance of two or more targetnucleic acids, the composition includes two or more promoter-basedoligomers each configured to selectively hybridize a cDNA containing thesubstantial complement their respective target nucleic acids, whereineach of the two or more promoter based oligomers comprise a tag sequencebetween their 5′ promoter regions and their 3′ a b c regions and whereineach of the tag sequences in each of these two or more promoter-basedoligomers are substantially identical. In some methods for detecting thepresence or abundance of two or more target nucleic acids, thecomposition includes two or more promoter-based oligomers eachconfigured to selectively hybridize a cDNA containing the substantialcomplement of their respective target nucleic acids, wherein each of thetwo or more promoter based oligomers comprise a tag sequence betweentheir 5′ promoter regions and their 3′ a b c regions and wherein each ofthe tag sequences in each of these two or more promoter-based oligomersare substantially identical and wherein the composition further includesan additional promoter based oligomer comprising a target hybridizingsequence that is also substantially identical to the tag sequences. Somecompositions include at least one promoter-based oligomer that is apromoter primer. Some compositions include at least one promoter-basedoligomer that is a promoter provider. Some methods herein includehybridizing a promoter-based oligomer to a nucleic acid sequence that issubstantially complementary to all or a portion of a target nucleic acidand providing conditions for generating an RNA amplification product.Some methods herein include hybridizing a promoter-based oligomer to anucleic acid sequence that is substantially complementary to all or aportion of a target nucleic acid in the presence of a target captureoligomer comprising a target hybridizing sequence that is substantiallycomplementary to a target nucleic acid and providing conditions forgenerating an RNA amplification product from the promoter-basedamplification product. Some methods herein include hybridizing apromoter-based oligomer to a nucleic acid sequence that is substantiallycomplementary to all or a portion of a target nucleic acid in thepresence of a target capture oligomer comprising a target hybridizingsequence that is substantially complementary to a target nucleic acid,wherein the target capture oligomer is in a hairpin form, and providingconditions for generating an RNA amplification product from thepromoter-based amplification product. Some methods herein includehybridizing a promoter-based oligomer to a nucleic acid sequence that issubstantially complementary to all or a portion of a target nucleic acidin the presence of a target capture oligomer comprising a targethybridizing sequence that is substantially complementary to at least aportion of the promoter-based oligomer target hybridizing sequence, andproviding conditions for generating an RNA amplification product fromthe promoter-based amplification product. Some methods herein includehybridizing a promoter-based oligomer to a nucleic acid sequence that issubstantially complementary to all or a portion of a target nucleic acidin the presence of a target capture oligomer comprising a targethybridizing sequence that is substantially complementary to at least aportion of the promoter-based oligomer target hybridizing sequence,wherein the target capture oligomer is in a hairpin form, and providingconditions for generating an RNA amplification product from thepromoter-based amplification product. Some methods herein includehybridizing a promoter-based oligomer to a nucleic acid sequence that issubstantially complementary to all or a portion of a miR-21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acid in the presenceof a target capture oligomer comprising a target hybridizing sequencethat is substantially complementary to at least a portion of thepromoter-based oligomer target hybridizing sequence, and providingconditions for generating an RNA amplification product from thepromoter-based amplification product. Some methods herein includehybridizing a promoter-based oligomer to a nucleic acid sequence that issubstantially complementary to all or a portion of a miR-21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acid in the presenceof a target capture oligomer comprising a target hybridizing sequencethat is substantially complementary to at least a portion of thepromoter-based oligomer target hybridizing sequence, wherein the targetcapture oligomer is in a hairpin form, and providing conditions forgenerating an RNA amplification product from the promoter-basedamplification product. Some methods herein include generating anamplification reaction mixture that comprises at least onepromoter-based oligomer and at least one target capture oligomer,wherein the promoter-based oligomer comprises a target hybridizingsequence that is substantially identical to all or a portion of amiR-21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleicacid sequence, and wherein the target capture oligomer comprises atarget hybridizing region that is substantially complementary to all ora portion of a miR-21, miR-34b, miR-182, miR-221, miR-222 or miR-802target nucleic acid sequence. Some methods herein include generating anamplification reaction mixture that comprises at least onepromoter-based oligomer, at least one target capture oligomer and atleast one first amplification oligomer, wherein the promoter-basedoligomer comprises a target hybridizing sequence that is substantiallyidentical to all or a portion of a miR-21, miR-34b, miR-182, miR-221,miR-222 or miR-802 target nucleic acid sequence, wherein the targetcapture oligomer comprises a target hybridizing region that issubstantially complementary to all or a portion of a miR-21, miR-34b,miR-182, miR-221, miR-222 or miR-802 target nucleic acid sequence, andwherein the first amplification oligomer comprises a target hybridizingregion that is substantially complementary to all or a portion of amiR-21, miR-34b, miR-182, miR-221, miR-222 or miR-802 target nucleicacid sequence. Some methods herein include performing an amplificationreaction in an amplification reaction mixture comprising amplificationoligomers and at least one target capture oligomer wherein the targetcapture oligomers are formed to a hairpin formation under a set ofconditions and wherein the amplification reaction proceeds at a set ofconditions configured to maintain the target capture oligomer as ahairpin.

Some methods include at least one detection probe oligomer forhybridizing to a target nucleic acid sequence. Some methods fordetecting the presence or abundance of two or more target nucleic acidscan use two or more detection probe oligomers each configured toselectively hybridize each of their respective target nucleic acids.Some detection probe oligomers for hybridizing to a target nucleic acidsequence are linear probes. Some detection probe oligomers are labeledlinear probes. Some detection probe oligomers are dual labeled linearprobes. Some detection probe oligomers are hairpin probes. Somedetection probe oligomers are labeled hairpin probe. Some detectionprobe oligomers are dual labeled hairpin probe. Some detection probeoligomers are molecular beacons. Some detection probe oligomers aremolecular torches. Some detection probe oligomers comprise a targethybridizing region that is substantially complementary to all or aportion of a micro RNA. Some detection probe oligomers comprise a targethybridizing region that is substantially complementary to a portion of amicro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotidesof the micro RNA. Some detection probe oligomers comprise a targethybridizing region that is substantially complementary to a portion of amicro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 contiguous nucleotidesat the 3′ end of the micro RNA. Some detection probe oligomers comprisea target hybridizing region that is substantially complementary to aportion of a micro RNA, wherein the portion is 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27contiguous nucleotides at the 5′ end of the micro RNA. Some detectionprobe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-21. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-34b. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-182. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-221. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-222. Somedetection probe oligomers comprise a target hybridizing region that issubstantially complementary to all or a portion of miR-802. Some methodsherein include at least one detection probe oligomer for hybridizing toan amplification product. Some methods herein for detecting the presenceor abundance of two or more target nucleic acids can use two or moredetection probe oligomers each configured to hybridize to anamplification product generated from the two or more target nucleicacids. Some methods herein for detecting the presence or abundance oftwo or more target nucleic acids can use two or more detection probeoligomers each configured to selectively hybridize to an amplificationproduct generated from the two or more target nucleic acids. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template using anamplification oligomer described herein. Some detection probe oligomersare configured to hybridize to an amplification product generated from amicro RNA template using an amplification oligomer selected from a firstamplification oligomer as described herein, a promoter-based oligomer asdescribed herein, a reverse primer oligomer as described herein orcombinations thereof. Some detection probe oligomers are configured tohybridize to an amplification product generated from a micro RNAtemplate using a first amplification oligomer in a 5′ Z.sub.n a′ b′ c′3′ format as described herein, wherein the detection probe oligomercomprises a target hybridizing sequence that is substantiallycomplementary to a portion of the extension nucleic acid sequence. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template using a firstamplification oligomer in a 5′ Z.sub.n a′ b′ c′ 3′ format as describedherein, wherein the detection probe oligomer comprises a targethybridizing region that is substantially identical to all or a portionof the micro RNA template. Some detection probe oligomers are configuredto hybridize to an amplification product generated from a micro RNAtemplate using a first amplification oligomer in a 5′ Z.sub.n a′ b′ c′3′ format as described herein, wherein the detection probe oligomercomprises a target hybridizing region that is substantiallycomplementary to a portion of the extension nucleic acid sequence and toall or a portion of the amplification product sequence that is itselfcomplementary to the micro RNA template. Some detection probe oligomersare configured to hybridize to an amplification product generated from amicro RNA template using a promoter-based oligomer as described herein,wherein the detection probe oligomer comprises a target hybridizingsequence that is substantially identical to all or a portion of thetarget hybridizing sequence of the promoter-based oligomer. Somedetection probe oligomers are configured to hybridize to anamplification product generated from a micro RNA template using apromoter-based oligomer containing a tag sequence as described herein,wherein the detection probe oligomer comprises a target hybridizingsequence that is substantially identical to all or a portion of thetarget hybridizing sequence of the promoter-based oligomer, all or aportion of the tag sequence or a combination thereof. Some detectionprobe oligomers are configured to hybridize to an amplification productgenerated from a micro RNA template using a promoter-based oligomer asdescribed herein, wherein the detection probe oligomer comprises atarget hybridizing region that is substantially complementary to an RNAtranscript amplification product.

Some methods herein use chemical compounds, such as enzymes, substrates,acids or bases to adjust pH of a mixture, salts, buffers, chelatingagents, denaturants, sample preparation agents, sample storage ortransport medium, cellular lysing agents, total RNA isolation componentsand reagents, partial generalized RNA isolation components and reagents,solid supports, and other inorganic or organic compounds. Methods mayinclude using any combination of the herein mentioned components andother components not mentioned herein. Reaction mixtures useful in themethods can be provided as part of a commercially available product.Reaction mixtures useful in the methods can be provided premixed orpartially premixed as part of a commercially available product. Reactionmixtures useful in the methods can be provided as separate components aspart of a commercially available product.

Aspects and embodiments of the present invention are illustrated in theExamples that follow. Methods and reagents for nucleic acid synthesis,hybridization, and detection of labels were used substantially asdescribed herein, although those skilled in the art will appreciate thatother routine methods and standard reagents may also be used to achieveequivalent results. Oligonucleotides were synthesized using standardphosphoramidite chemistry (Caruthers et al., 1987, Methods in Enzymol.,154: -287), purified using routine chromatographic methods (e.g., HPLC),and typically stored in a solution of 10 mM Tris, 1 mM EDTA (pH 7.5), atroom temperature to −80.deg.C. In the target capture steps illustratedin the examples, magnetic particles were used as the capture supportwhich were separated from the soluble phase by applying a magnetic fieldto the outside of the assay container, although those skilled in the artwill appreciate that other means of separation may be used. Thesupernatant containing soluble components was removed, and thehybridization complexes bound to the particles were washed (one to threetimes with a washing solution of sufficient ionic strength to maintainbonds binding the captured hybrid to the magnetic particles at thewashing temperature, usually about 25.deg.C.). Washing generally isperformed at room temperature by suspending the particles in the washingsolution, separating particles, and removing the supernatant, andrepeating those steps for each wash. For the detection step, thedetection probe was incubated with the released target nucleic acid inan aqueous solution containing appropriate salts and buffers at atemperature below the Tm predicted for the detection probe sequence andits target sequence, usually for 30-60 min. When an AB-labeled detectionprobe was used, a homogeneous detection step was performed which usesdifferential hydrolysis of the AE label on unbound probes compared toAE-labeled probes bound to the target (described in detail in U.S. Pat.No. 5,283,174). For example, hydrolysis was performed by adding aselection reagent that promotes hydrolysis of the AE label on unboundprobes (e.g., a basic solution), followed by adding a detection reagentthat catalyzes chemiluminescence from AE attached to bound probes (e.g.,H.sub.20.sub.2), and the chemiluminescent signal (referred to asrelative light units or RLU) was detected on a luminometer (e.g.,LEADER® 450HC+, Gen-Probe Incorporated, San Diego, Calif.).

The following examples describe some preferred embodiments and reagentsused in these assays. The skilled artisan will appreciate that otherreagents and conditions may be substituted for those described herein toperform the method steps. For example, the reagents and conditions forproducing and detecting a signal will be selected by the skilled artisanbased on the chosen detection probe label. Those skilled in the art willunderstand that the invention methods may be performed using any chosentarget nucleic acid sequence that can hybridize to a complementarysequence, i.e., the method is not dependent on any particular probe ortarget sequences. One of ordinary skill in the art will be able toselect the target sequence and then design and synthesize theappropriate target hybridizing sequence of any of the capture probeforms described herein, and a target-specific detection probe by usingroutine methods. That is, specific assays will rely on selection of atarget sequence and the appropriate target hybridizing sequencescontained in the capture and detection probes that include thestructural characteristics described herein, and such selection can beperformed by one of ordinary skill in the art using standard procedures,followed by routine testing of the designed components to optimizedetection of the selected target by using the methods described herein.

Example 1 Detection of a Small Target Nucleic Acid using DifferentLabeled Probes

To design detection probes, a target sequence of 23 nt was selected froma sequence common to genomic sequences of human Herpesvirus 5(Cytomegalovirus) strains (Dunn et al., 2003, Proc. Natl. Acad. Sci. USA100(24): 14223-14228; GenBank accession nos. AC146999, AC146851, andAY315197) and complementary to portions of fluorescent protein genes(GenBank accession nos. AY 303166, AY303167, and AY237157). Oligomers of23 nt that were completely complementary to the target sequence weresynthesized in vitro as a 2′-O-methyl oligoribonucleotides that had 52%GC content. Three versions of the probes were labeled with a linker atdifferent positions (between bases 8 and 9, 12 and 13, and 13 and 14 ofthe 23-mer) and an AE label attached at the linker by using methodspreviously described in detail (U.S. Pat. Nos. 5,185,439, 5,283,174, and5,656,744). The labeled probes (0.1 pmol per reaction) were individuallyhybridized at 60.deg.C. for 1 hr to a complementary synthetic ssRNAtarget sequence (10 pmol per reaction) in a 30 .micro.1 reaction mixturecontaining 15 .micro.1 of a hybridization reagent (190 mM succinate, 17%(w/v) lithium lauryl sulfate (LLS), 100 mM LiOH, 3 mM EDTA, and 3 mMEGTA, at pH 5.1). Then the hybridization mixture was diluted to 500.micro.1 with the hybridization reagent and 20 .micro.1 aliquots wereremoved for performing the detection step by adding to each detectionreaction mixture 80 .micro.1 of the hybridization reagent, and then 200.micro.1 of a selection reagent (600 mM boric acid, 182.5 mM NaOH, 1%(v/v) octoxynol (TRITON® X-100), at pH 8.5), and hydrolysis wasperformed at 50.deg.C. for varying periods of time. Then, production anddetection of the signal was performed by adding 200 ,micro.1 of adetection reagent (1 mM nitric acid and 32 mM H.sub.2O.sub.2) followedby adding 200 .micro.1 of 1.5 M NaOH and the chemiluminescent signal(RLU) was measured (for 2 sec) by using a luminometer. The samedetection reaction method was performed on aliquots that contained theindividual probes without the ssRNA target to measure hydrolysis of theAE label on unbound probes. From these results, the time at which halfof the detectable label for each probe composition was hydrolyzed(T1/2), when the complementary target strand was present or absent, wasdetermined. The T1/2 for all three probes without the target (i.e.,unbound probes) was between 0.69 and 1.05 min, whereas when the targetwas present (i.e., bound probes) the T1/2 was between 25.8 and 125 min,indicating that the detection probes bound to the target and produced adetectable signal in excess of the background signal in a homogeneousreaction mixture. When probes were hybridized with the target, theyexhibited different T1/2 characteristics: the probe labeled betweenpositions 12 and 13 had the shortest T1/2 (25.8 min), the probe labeledbetween positions 13 and 14 had the longest T1/2 (125 min), and theprobe labeled between positions 8 and 9 was an intermediate T1/2 (69min). These results show that all three probes were capable of bindingto the complementary RNA target, that labels in unbound probes could bedistinguished from labels in bound probes by differential hydrolysischaracteristics, and that the labeling position on the oligomer affectedthe rate of label hydrolysis so that optimal probes for an assay may beselected and designed using routine testing.

Example 2 Sensitivity of Detection of Single-Stranded andDouble-Stranded Targets

The sensitivity of target detection was determined by using the sametarget and detection probe sequences as in Example 1, but comparingdetection of the RNA target when it was in ssRNA or dsRNA form. ThessRNA target oligomer and detection probe oligomer labeled with AEbetween positions 13 and 14 were used as in Example 1. In these assays,all reactions contained 0.1 pmol of the AE-labeled probe that washybridized to the ssRNA target present in a range of 0 to 5 fmol perhybridization reaction (100 .micro.1 hybridization mixtures incubated at60.deg.C. for 1 hr). Following hybridization, the AE label on unboundprobe was hydrolyzed by adding 200 .micro.1 of the selection reagent andincubating at 50.deg.C. for 10 min, and then the chemiluminescent signalfrom bound probe was detected substantially as described in Example 1.Results shown in Table 1, columns 1 and 2, demonstrate that a lineardetectable signal was measured over the range of target amounts tested.As little as 0.005 fmol of the ssRNA target in the reaction resulted ina detectable signal over the background signal obtained when no targetnucleic acid was present in the assay. “Net RLU” data (column 2) wascalculated by subtracting the background RLU (560 RLU when no target waspresent) from the detected RLU for each test sample.

In the tests performed using the dsRNA target, the target was made bysynthesizing a complementary RNA strand to the ssRNA target oligomerdescribed above and hybridizing the two complementary RNA strandstogether. The dsRNA target was tested substantially as described aboveby using the same probe as described above synthesized as a 2′-O-methyloligoribonucleotide and labeled with AE between positions 13 and 14.This detection probe was complementary to one of the strands in thedsRNA target. Before hybridization with the AE-labeled probe, the dsRNAtarget was denatured by heating it in solution (10 mM Li-succinate and0.01% LLS, pH5.0) at 90.deg.C. for 5-7 min, followed by quickly coolingon ice. In Test 1, 50 pmol of the target dsRNA was denatured and thendiluted to make the different amounts of target used in each of the 100.micro.1 hybridization reactions. In Test 2, the appropriate amounts ofthe target dsRNA were distributed to separate tubes in 50 .micro.1aliquots, heat denatured as described above, and then 50 .micro.1 of thehybridization reagent containing the labeled probe was added to eachtube make the hybridization reaction mixtures. The hybridization anddetection reactions were performed substantially as described above forthe ssRNA target reactions and the results for the dsRNA target areshown in Table 1, columns 3 to 5. The background signal detected when notarget was present (942 RLU in Test 1, 932 RLU in Test 2) was subtractedfrom the detected signal when the dsRNA target was present to obtain the“Net RLU” (column 4 for Test 1, and column 5 for Test 2). The Net RLUmeasurements showed that the assay produced a detectable signal that wasa substantially linear response over the range of target amounts tested.A positive signal was detected when as little as 0.01 fmol (Test 1) to0.05 fmol (Test 2) of the target RNA was present in the reactionindicating the sensitivity of the detection step.

TABLE 1 Signal Measured for Hybridization Reactions Containing DifferentAmounts of Target ssRNA dsRNA Net RLU - Net RLU - (fmol) Net RLU (fmol)Test 1 Test 2 0.005 391 — — — 0.01 719 0.01 122 — 0.02 1,289 0.02 151 —0.05 3,376 0.05 625 2,796 0.07 4,648 0.07 567 — 0.1 7,157 0.1 1,0665,561 0.25 16,922 0.25 2,079 — 0.5 29,729 0.5 4,443 27,142 1.0 64,9291.0 7,974 — 5.0 287,821 5.0 54,077 240,787

Example 3 Capture and Detection of a Target RNA

In these assays, a capture probe capable of forming a hairpin structureunder hybridization conditions was used to capture a target nucleic acidfrom a sample, followed by hybridization of the target nucleic acid witha labeled detection probe and detection of a signal from bound detectionprobe. The capture probes used in these experiments all containstructural features that allow formation of a hairpin structure: a 5′region homopolymeric sequence, a 3′ region sequence that was fully orpartially complementary to the 5′ region sequence, and a targethybridizing sequence flanked by the 5′ and 3′ region sequences. The 5′and 3′ region sequences form the “stem” portion of the hairpinstructure, and the target hybridizing sequence forms the “loop” portionof the hairpin structure.

Three versions of a hairpin capture probe were synthesized and assayedusing routine methods to determine the Tm of the stem of the hairpincapture probe. The complementary 5′ and 3′ region sequences of all threeprobes were synthesized with deoxyribonucleotide linkages. The targethybridizing sequence of each of the hairpin probes was the 23-nt targethybridizing sequence as in Example 1, synthesized in probes 1 and 2 with2′-O-methyl linkages and in probe 3 with deoxyribonucleotide linkages.In probes 1 and 3, the 5′ region was a (dT)12 sequence and the 3′ regionwas a (dA)12 sequence; and in probe 2, the 5′ region sequence was(dT)5A(dT)6 which is partially complementary to the 3′ region sequenceof (dA)12. Schematically, the resulting capture probe sequences were asfollows: 5′ TTTTTTTTTTTT—N.sub.23—AAAAAAAAAAAA 3′ (probes 1 and 3; SEQID NO:1), and 5′ TTTTTTATTTTTT—N. sub.23—AAAAAAAAAAAA 3′ (probe 2; SEQID NO:2), in which N₂₃ represents the target hybridizing sequence. Inwill readily be appreciated that these linear sequences form partiallydouble-stranded hairpin structures by intramolecular hybridization ofthe 5′ region to the 3′ region and the target hybridizing sequence(N.sub.23) becomes the loop portion of the hairpin structure. The Tm'sfor the double-stranded stem portions of these hairpin probes were in arange of about 46.deg.C. to about 57.deg.C. (46.3.deg.C. for probe 2,55.7.deg.C. for probe 3, and 56.7.deg.C. for probe 1).

Capture using these hairpin capture probes and detection of the targetwas performed using the dsRNA target and AE-labeled probe described inExample 2, using target amounts ranging from 0.05 to 5 fmol perreaction. To provide a sample similar to a clinical sample, the dsRNAtarget present in 200 .micro.1 of sample transport solution (110 mM LLS,15 mM sodium phosphate monobasic, 15 mM sodium phosphate dibasic, 1 mMEDTA, 1 mM EGTA, pH 6.7) was mixed with 200 .micro.1 of urine, to make afinal sample volume of 400 .micro.1. This mixture was heated to denaturethe dsRNA target (at 90.deg.C. for 5 min, then cooled on ice), toprovide a ssRNA target strand for hybridization with the capture probes.For each of the hairpin capture probes tested individually, thedenatured RNA target sample was mixed with 100 .micro.1 of a targetcapture reagent (250 mM HEPES, 310 mM LiOH, 1.88 M LiCl, 100 mM EDTA, pH6.4) containing 0.3 pmol of the hairpin capture probe and 50 .micro.g ofmagnetic particles which were the capture support (1 micron Sera-MagTMMG-CM particles, Seradyn, Inc. Indianapolis, Ind.), to which immobilizedprobe oligomers of dT.sub.14 were covalently attached. The mixture wasincubated at 65.deg.C. for 60 min (a temperature above the Tm of each ofthe capture probes) and then at room temperature for 30 min to formcapture hybrids attached to the particles. Then, the particles with theattached capture hybrids were separated magnetically from the liquidsample components that were removed. The particles with attached capturehybrids were washed twice at room temperature with 500 .micro.1 of thesample transport solution and then the particles with attached capturehybrids were separated from the solution which was removed. The washedparticles with the attached capture hybrids were mixed with 100 .micro.1of water and heated (90.deg.C. for 5 min) to release the nucleic acidcomponents of the capture hybrids (target and capture probe oligomersreleased into solution and the immobilized probe remained covalentlyattached to the capture support particle). For detection of the target,the solution then was mixed with an AB-labeled detection probe, asdescribed in Example 2, in 100 .micro.1 of the hybridization reagent andthe mixture was incubated under hybridization conditions (55.deg.C. for60 min) to allow the detection probe to bind to the target strands.Under these conditions, the released capture probes may reform thepartially double-stranded hairpin structure by intramolecularhybridization to minimize competitive inhibition caused by the captureprobes competing with the detection probes for hybridization to thetarget strand. The detection probe and the target hybridizing sequenceof the hairpin capture probes will not hybridize to each other becausethey are the same sense strands. Detection of the signal from bounddetection probes was performed substantially as described in Example 1.Control reactions without target were treated identically and thebackground signal for all reactions was in the range of 535 to 715 RLU.The experimental results of these assays are shown in Table 2, column 2,as net RLU (detected RLU minus background RLU). For each assay, theratio of the detected signal to background RLU is shown in Table 2,column 3.

TABLE 2 Assays Performed Using a Hairpin Capture Probe and DetectionProbe Target Amount Signal/Background (fmol) Net RLU Ratio 0.05 1,248 30.1 2,223 4.4 0.15 3,318 6.6 0.2 5,418 9 0.5 11,156 22 1.0 24,758 36 2.038,351 55 5.0 98,180 140The results of these assays show that the combination of capture of atarget nucleic acid by using a hairpin capture probe and detection byusing a detection probe complementary to one strand of a dsRNA targeteffectively detected the target present in a sample for all of theamounts of target tested.

Example 4 Assays Comparing Different Forms of Capture Probes

These assays compared the relative efficiency of capture and detectionof a target sequence, using methods similar to those described inExample 3, when the target capture step was performed by using a captureprobe of either a hairpin structure or linear structure. Unlessotherwise stated, the reagents used in these tests were the same asdisclosed in Examples 1 to 3 above. All of the assays used test samplescontaining 1 fmol of the ssRNA target, as described in Example 2, in 200.micro.1 of urine mixed with 200 .micro.1 of sample transport solution.For the target capture step, each 400 .micro.1 test sample was mixedwith 100 .micro.1 of target capture reagent containing different amounts(0.1, 0.5, 1.0, 2.0, 5.0, 10 and 20 pmoles) of either a partiallydouble-stranded hairpin capture probe as described in Example 3 (SEQ IDNO:1) or a linear single-stranded capture probe of the followingstructure: 5 ′ X N.sub.23 TTTAAAAAAAAAAAA 3′ (SEQ ID N0:3) that hassubstantially the same target hybridizing sequence (N.sub.23) as in thehairpin capture probe, but includes one additional 5′ nucleotide (X). Inboth the hairpin and linear forms of the capture probes, the targethybridizing sequences were synthesized with 2′-O-methyl linkages. In thehairpin capture probe, the target hybridizing sequence was flanked bythe complementary 5′ poly-dT and 3′poly-dA regions, whereas in thelinear form the target hybridizing sequence was covalently linked to a3′ (dT).sub.3(dA).sub.30 sequence. The reaction mixtures were incubatedat 65.deg.C. for 60 min and then at room temperature for 30 min to allowformation of capture hybrids and attachment to the capture support viathe immobilized probe. The supports with attached capture hybrids wereseparated from the liquid sample components by applying a magnetic fieldand washed twice (using 0.5 ml of sample transport solution each)substantially as described in Example 3. The final wash solution wasremoved and the capture supports with attached capture hybrids weremixed with 100 .micro.1 of water per assay, incubated at 90.deg.C. for 5min and rapidly cooled on ice to release the capture hybrids into thenucleic acid components before hybridization of the target strand withthe detection probe. Then, each test sample was mixed with 0.1 pmole ofthe AE-labeled detection probe of Example 2 in 100 .micro.1 ofhybridization reagent and incubated at 55.deg.C. for 60 min to allowhybridization of the detection probe to the target strand. Detection ofthe chemiluminescent signal from detection probes bound to the targetstrands was performed substantially as described in Example 3 (add 200.micro.1 of selection reagent, incubate at 55.deg.C. for 10 min, mixwith 200.micro.1 of detection reagent and measure chemiluminescence (for5 sec) on a luminometer). The results are shown in Table 3, as net RLUin column 2 and 3, and the relative percentage of detection obtainedwhen the capture step had been performed by using the linear or hairpinforms of the capture probes, in columns 4 and 5. The net RLU wascalculated by subtracting the background RLU from the RLU detected inpositive samples (background was 762 RLU for the hairpin probe tests and749 RLU for the linear probe tests). The relative percentage ofdetection was calculated by setting the highest detected net RLU at 100%(results for 0.1 pmole of hairpin capture probe) and dividing the lessernet RLU detected in the other tests by the highest net RLU (21,778).

TABLE 3 Comparison of Hairpin and Linear Capture Probes Capture ProbeNet RLU Net RLU % Detection % Detection (pmole) Hairpin Probe LinearProbe Hairpin Probe Linear Probe 0.1 21,778 15,849 100 72.8 0.5 19,6147,286 90.1 33.5 1.0 18,248 4,172 83.8 19.2 2.0 17,316 2,578 79.5 11.85.0 11,889 1,137 54.6 5.2 10 9,778 722 44.9 3.3 20 4,519 458 20.7 2.1

These results show that the assays performed by using a linear form anda hairpin form of the capture probes specific for the same targetsequence resulted in a detectable signal for all of the assaysperformed. The relative percentage of detection was consistently higherwhen the capture probe was in the hairpin form compared to the linearform. The difference in relative percentage of detection ranged fromabout 10-fold more when the results obtained for the two forms werecompared for the highest amounts of capture probes tested (20 pmoles perreaction), to about 2.7-fold when the results obtained for the two formswere compared for the lowest amount of capture probes tested (0.1 pmoleper reaction). The differences between the assays that used the hairpinand linear capture probe forms may result from more competitiveinhibition when the linear capture probe was used because the releasedlinear form may compete with the detection probe for hybridization tothe target sequence during the detection phase of the assay whereas thehairpin form under the same conditions may reform the hairpin structureto limit competition between the target hybridizing sequence of thecapture probe and the detection probe for binding the target.

Example 5 Assays Using a Partially Double-Stranded Capture Probe

This example describes an embodiment that uses a completely or partiallydouble-stranded capture probe. The capture probe of this embodiment ismade up of two completely or partially complementary strands of whichone strand includes a target hybridizing sequence that binds to aportion of the target nucleic acid. One version of the capture probe ismade up of a first capture probe strand (SEQ ID NO:4) and a secondcapture probe strand (SEQ ID NO:5) that are synthesized and hybridizedtogether to make a partially double-stranded capture probe bound byhybridization of at least their complementary 3′ polyA and 5′ polyTsequences: 5′TTTTTTTTTTTTTTTAGAGGATGGGTTTTCTAGGGG 3′ (SEQ ID NO:4),3′AAAAAAAAAAAAAAATCTCTCTCTCTCTCTCTCTC 5′ (SEQ ID NO:5). The oligomer ofSEQ ID NO:4 contains a 5′ poly-T region and a 3′ sequence complementaryto a sequence contained in a human B19 parvovirus genome (GenBankaccession no. AY386330); and the oligomer of SEQ ID NO:5 contains a 5′poly-(TC) region and a 3′ poly-A region. In another version, the captureprobe is completely double stranded and made up of the first captureprobe strand (SEQ ID NO:4) hybridized to its complementary strand (SEQID NO:6): 5′TTTTTTTTTTTTTTTAGAGGATGGGTTTTCTAGGGG 3′ (SEQ ID NO:4),3′AAAAAAAAAAAAAAATCTCCTACCCAAAAGATCCCC 5′ (SEQ ID NO:6).

In separate assays, about 3.5 pmole of each version of the captureprobes is mixed with a 0.5 ml plasma sample containing parvovirus B19genomic DNA (denatured and, optionally, sheared or enzymaticallydigested into fragments of about 100 to 1000 lit long) and an equalvolume of target capture reagent containing capture support particleswith attached poly(A) oligomers as the immobilized probe. That is, theimmobilized poly(A) oligomers are complementary to the 5′ poly-dTportion of the capture probe oligomer of SEQ ID NO:4. The mixture isincubated (60-65.deg.C., 20-60 min) to allow the capture probes todissociate into the component oligomers (SEQ ID NO:4 and SEQ ID NO:5, orSEQ ID NO:4 and SEQ ID NO:6), to allow the target-specific portion ofSEQ ID NO:4 to hybridize to the complementary sequence in the parvovirusB19 target DNA. Then, the mixture is incubated at a lower temperature(25-30.deg.C., 14-30 min) to allow the complementary homopolymericsequences of the capture probe and the immobilized probe to hybridize,thereby attaching the target B19 DNA to the magnetic particles in acapture hybrid that includes the capture probe strands of SEQ ID NO:5hybridized to the complementary sequence in the parvovirus B19 DNA.Particles with the attached capture hybrids are separated from thesample components by applying a magnetic force to the outside of thecontainer and the liquid sample components, including unhybridizedcapture probe strands, are removed. A washing step is used in which theparticles with the attached capture hybrids are suspended in an aqueoussolution of sufficient ionic strength the maintain the capture hybridattached to the capture support, then the particles are separated fromthe aqueous solution using magnetic force, and the washing solution withany unbound capture probe oligomers and other sample components isremoved. For detection of the captured B19 DNA, the particles with theattached capture hybrids are mixed with a hybridization reagentcontaining detection probe oligomers of SEQ ID NO:7 (0.1-0.5 pmoles perreaction) labeled with a fluorescent label (e.g., fluorescein) and thedetection probes are hybridized to a complementary sequence in thecaptured B19 DNA by incubating the mixture below the Tm of the detectionprobe but at a temperature above the melting temperature of thepolA-polyT duplex, to release the B19 target nucleic acid into thesolution phase (e.g., 55.deg.C. for 20-60 min). The mixture is thenincubated at a lower temperature (e.g., 25-30.deg.C. for 10-30 min) toallow hybridization complexes made up of the B19 target DNA, detectionprobe and the poly-dT containing capture probe strand to attached to thepoly (A) immobilized probes on the particles. The particles withattached complexes that contain the hybridized detection probes areseparated from the solution phase as described above and the solutionphase containing unbound detection probes is discarded. The particleswith attached complexes are optionally washed, substantially asdescribed above, under conditions that maintain the hybridizationcomplexes on the particles to remove remaining unbound detection probes.Finally, the particles are mixed with a volume of liquid (e.g., 0.5 mlwater) and the fluorescence of the mixture is measured using standardfluorometric procedures. A negative control sample, i.e. plasmacontaining no B19 particles or DNA, is treated identically as describedabove and the fluorescence that is measured from the negative controlsample indicates background signal for the assay. For both the partiallyand completely double-stranded versions of the capture probe, theseassays produce a detectable positive signal for test samples thatcontain parvovirus B19 nucleic acid that is significantly greater thanthe background signal that negative control samples produce. Positivesignals indicate the presence of the parvovirus B19 target nucleic acidin the samples.

Example 6 Specific Capture, Amplification and Detection of miRNABiomarkers

Materials and Methods used in the following examples. Cell lines andxenografts: LNCaP, DU145, PC-3 and VCaP prostate cancer lines wereobtained from the American Type Culture Collection (Manassas, Va.).LNCaP and DU145 cell lines were cultured in RPMI Medium 1640 Custom(Invitrogen, Carlsbad, Calif.) containing 10% FBS. The PC-3 cell linewas cultured in MEM with Earle's salts (Invitrogen) supplemented with10% FBS, 2 mM L-glutamine, and 0.9 mM sodium pyruvate. VCaP cells werecultured in DMEM containing 10% FBS. Freshly frozen samples of 21 humanprostate cancer severe combined immunodeficient xenografts (LuCaPs23.12, 23.1, 23.1AI, 35, 35V, 49, 70, 77, 78, 81, 86.2, 92, 93, 96,96AI, 105, 115, 141, 145.1, 145.2, 153) were prepared using standardmethods. Characterization of several of these xenografts has beenreported previously (Laitinen, Genes Chromosomes Cancer 2002, 35:66-73;Saramaki, Int J Cancer 2006, 119:1322-1329; Porkka, Cancer Res 2007,67:6130-6135). Normal prostate tissue samples: Five adjacent-normalprostate tissues, pathologically determined to be 100% normal(OCT-embedded), were purchased from Cytomyx Inc. (Lexington, Mass.).These samples were shipped to Asuragen Services (Austin, Tex.) forsubsequent RNA extraction and microRNA profiling.

RNA samples: Total RNA was extracted from cell lines using the AmbionmirVana miRNA isolation kit (Applied Biosystems Inc., Foster City,Calif.) and frozen sections of xenograft tissue using Trizol reagent(Invitrogen) according to the manufacturer's instructions. Syntheticmature miRNAs were purchased from Integrated DNA Technologies(Coralville, Iowa). The human pre-miR-802 sequence was obtained from themiRBase (Griffiths-Jones, Nucleic Acids Res 2006, 34:D140-4) of theSanger Institute and a synthetic hairpin precursor was synthesized usingPCR amplification of the 94 nt pre-miR-802 sequence from LNCaP genomicDNA cloned into pBluescript II SK (+) and expressed as an in vitrotranscript.

Real-time quantitative PCR: cDNA was prepared from synthetic maturemiRNAs or total RNA using specific TaqMan Assays-on-Demand reversetranscription primers and TaqMan miRNA Reverse Transcription Kit(Applied Biosystems Inc.). Absolute copy number of mature miRNAs wasdetermined by qRT-PCR using TaqMan Assays-on-Demand primer and probesets along with TaqMan Universal PCR master mix (Applied BiosystemsInc.) for cDNA amplification. Amplification and analysis were performedon the ABI 7000 sequence detection system. Copies per cell weredetermined from total nanograms of RNA using an estimated 15 picogramsof total RNA per cell as described (Chen, Nucleic Acids Res 2005,33:e179).

Real-time transcription-mediated amplification: All buffer and enzymereagents used in the real-time TMA assays were APTIMA® reagents fromGen-Probe Incorporated (San Diego, Calif.). All reactions were run intriplicate. Amplification reactions were prepared in 96-well microtiterplates containing specifically designed T7-provider (U.S. Pat. No.7,374,885) and 3′-extender oligonucleotides together with a commonreverse primer and molecular beacon. The plates were transferred to anEppendorf Thermomixer R.sup.TM instrument and incubated at 42.deg.C. for5 minutes. Next, APTIMA enzyme reagent was rapidly pipetted into eachwell and a sealing card was applied to the plate. After a brief mixingstep (1 minute at 42.deg.C.), the plate was transferred to an MJ Chromo4instrument (Bio-Rad, Hercules, Calif.) that had been pre-heated to42.deg.C. Fluorescence readings were taken every 20 seconds at 42.deg.C.for 60 minutes. Emergence times were compared against calibrationstandards to derive miRNA copy numbers. The T7 provider oligonucleotidesused in these examples each contained a 3′-(reverse polarity)-dCnucleotide (5′-5′-phosphdiester linkage) to block 3′-extension (U.S.Pat. No. 7,374,885). Real-time TMA assays used the followingoligonucleotides for the different miRNAs, Table 4

TABLE 4 Promoter-Based Amplification Oligomer and Extender OligomerCombinations Small RNA T7 Provider (5′ → 3′) 3′ Extender (5′ → 3′)miR-21 AATTTAATACGACTCACTATA CGGTCGCAGAGATTAACTGGTACAGGGTGGGAGAUAGCUUAUCAGA TAAGCGTGGTCGACCGTCAACATCAGT miR-34bAATTTAATACGACTCACTATA CGGTCGCAGAGATTAACTGGTACAGGGT GGGAGATAGGCAGTGTCATAAGCGTGGTCGACCGCAATCAGCTAAT miR-182 AATTTAATACGACTCACTATACGGTCGCAGAGATTAACTGGTACAGGGT GGGAGATTTGGCAATGGTTAAGCGTGGTCGACCGAGTGTGAGTTCT miR-221 AATTTAATACGACTCACTATACGGTCGCAGAGATTAACTGGTACAGGGT GGGAGACCACAACGGTTTAGCTAAGCGTGGTCGACCGGAAACCCAGCAG UACAUUGUCUG miR-222 AATTTAATACGACTCACTATACGGTCGCAGAGATTAACTGGTACAGGGT GGGAGAAGCUACAUCUGGTAAGCGTGGTCGACCGGAGACCCAGTAG miR-802 AATTTAATACGACTCACTATACGGTCGCAGAGATTAACTGGTACAGGGT GGGAGACAGTAACAAAGATAAGCGTGGTCGACCGACAAGGATGAAT

All assays used the same reverse primer (5′-CGGUCGCAGAGATTAACT) andmolecular beacon labeled at the 5′ end with FAM and at the 3′ end withDabcyl (5′-CCGACAAGCGUGGUCGACGUCGG). Specific target capture of miRNAs:Capture of mature miRNAs was performed using the following chimerichairpin target capture oligonucleotides (TCO) (US Pat. Pub. No.2006/0068417): for miR-21(5′-TTTTTTTTTTTTUCAACAUCAGUCUGAUAAGCUAAAAAAAAAAAAA), for miR-182(5′-TTTTTTTTTTTTAGUGUGAGUUCUACCAUUGCCAAAAAAAAAAAAAAA), for miR-221(5′-TTTTTTTTTTTTGAAACCCAGCAGACAAUGUAGCUAAAAAAAAAAAA), for miR-222(5′-TTTTTTTTTTTTACCCAGUAGCCAGAUGUAGCUAAAAAAAAAAAA), and for miR-802 (5-TTTTTTTTTTTTACAAGGAUGAAUCUUUGUUACUGAAAAAAAAAAAA). The TCO was addedwith APTIMA Target Capture Reagent to RNA samples in Solution TransportMedium (Gen-Probe Inc.) and heated to 75.deg.C. for 15 minutes in a96-well deep well heater (model IC25 with block 620-5036, Torrey PinesScientific Inc., San Marcos, Calif.) to denature the hairpin. Thereaction mixture was then cooled to room temperature over 30 minutes toanneal each specific miRNA to its designated TCO. After binding to themiRNA, the 3′ polyA tail of the TCO was hybridized to the polyT tail ofa polyT derivatized magnetic beads (APTIMA Target Capture Reagent). Thebeads were collected using a Kingfisher96 PCR tip head (ThermoScientific, Waltham, Mass.), washed in APTIMA wash solution, and mixedwith APTIMA amplification reagent containing amplification and detectionoligonucleotides in a 96-well PCR plate as described above. To liberatethe miRNA from the TCO, the PCR plate was incubated at 90.deg.C. for 5minutes and then immediately cooled on ice for 5 minutes.

MicroRNA profiling: Total RNA was extracted from 22 human prostatecancer xenografts using Trizol Reagent (Invitrogen, Carlsbad, Calif.)with homogenization by Polytron. The samples were shipped to AsuragenServices for microRNA profiling. Samples for microRNA profiling studieswere processed by Asuragen Services according to the company's standardoperating procedures. Briefly, a custom-manufactured Affymetrix GeneChipfrom Ambion was designed to microRNA probes derived from Sanger mirBase9.2 and published reports (Cummins, Proc Natl Acad Sci USA 2006,103:3687-3692; Bentwich, Nat Genet 2005, 37:766-770; Berezikov, Cell2005, 120:21-24; Xie, Nature 2005, 434:338-345). The array was designedwith 2 staggered probes to represent each microRNA. The signalprocessing implemented for the Ambion miRCHIP is a multi-step processinvolving probe specific signal detection calls, background estimate andcorrection, constant variance stabilization, and either array scaling orglobal normalization. For each probe, an estimated background value issubtracted that is derived from the median signal of a set ofG-C-matched anti-genomic controls. Arrays within a specific analysisexperiment were normalized together according to the variancestabilization method (Huber, Bioinformatics 2002, 18 Suppl 1:S96-104).Detection calls were based on a Wilcoxon rank-sum test of the microRNAprobe signal compared to the distribution of signals from GC-contentmatched anti-genomic probes. For statistical hypothesis testing, atwo-sample t-Test with assumption of equal variance was applied. One-wayANOVA was used for experimental designs with more than two experimentalgroupings or levels of the same factor. These tests define which probesare considered to be significantly differentially expressed, orsignificant, based on a default p-value of 0.001 and log2 difference>1.

TaqMan validation of microarray results for normal and xenograft formiR-21, miR-182, miR-221 and miR-222: Absolute copy number of maturemicroRNAs was determined by quantitative real-time PCR (qRT-PCR) usingAssays-on-Demand TaqMan primer/probe sets along with TaqMan UniversalPCR master mix (Applied Biosystems Inc., Foster City, Calif.) for cDNAamplification. Amplification and analysis were performed on the ABI 7000sequence detection system. Copies per cell were determined using anestimated 15 pg total RNA per cell as described (Chen, Nucleic Acids Res2005, 33:e179).

Example 7 Real-Time TMA Assays Distinguish Mature miRNA Sequences fromPre-miRNA Sequences and from Related and Unrelated Non-Target miRNASequences

The miR-802 resides in chromosomal region 21q22.12, which is nearregions of TMPRSS2 and Ets family member chromosomal breakpoints(21q21.2-21.3) associated with aggressive forms of prostate cancer(Kumar-Sinha, Nat Rev Cancer 2008, 8:497-511; Mehra, Cancer Res 2008,68:3584-3590). A real-time TMA assay was performed for specificdetection of mature miR-802 template when compared to unrelated miRNAslet-7d, miR-34b and miR-548d and compared to the pre-miR-802 precursor(FIGS. 4A-C). Target nucleic acids were as follows: SEQ ID NO:34, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32, and SEQ ID NO:33, respectively.Target nucleic acids were prepared at 5×10.sup.7 copies and 10-foldserial dilutions down to 5 copies was made. Negative controls werewater. Amplification reactions were preformed in triplicate and includeone of the target or non-target nucleic acids. Amplification anddetection oligomers were SEQ IS NOS:13, 19, 20 and 21. Results arepresented in table 5 and in FIG. 4.

TABLE 5 Average TTime Nucleic Acid (amount) (minutes) let7d (5 copies)57.0 let7d (50 copies) 56.1 let7d (500 copies) 54.6 let7d(5,000 copies)55.4 let7d (50,000 copies) 56.4 let7d (5 × 10.sup5) 56.3 let7d (5 ×10.sup6) 59.2 let7d (5 × 10.sup7) 52.6 mir34b (5 copies) 55.6 mir34b (50copies) >60 mir34b (500 copies) >60 mir34b(5,000 copies) 58.4 mir34b(50,000 copies) >60 mir34b (5 × 10.sup5) 55.7 mir34b (5 × 10.sup6) 50.9mir34b (5 × 10.sup7) 51.7 mir548d (5 copies) 57.3 mir548d (50 copies)57.8 mir548d (500 copies) 57.1 mir548d(5,000 copies) 55.9 mir548d(50,000 56.2 copies) mir548d (5 × 10.sup5) 56.4 mir548d (5 × 10.sup6)54.0 mir548d (5 × 10.sup7) 51.0 mir802 (5 copies) 52.3 mir802 (50copies) 46.7 mir802 (500 copies) 39.6 mir802(5,000 copies) 35.7 mir802(50,000 copies) 31.1 mir802 (5 × 10.sup5) 27.6 mir802 (5 × 10.sup6) 23.4mir802 (5 × 10.sup7) 20.0

Some nonspecific amplification was observed for the let-7d and miR-34btargets after a threshold time (TTime) of 50 minutes (FIG. 4B) and incontrol reactions that contained no template. TTimes for miR-802 rangedfrom about 20 minutes to about 52 minutes, and detected as few as 5copies per reaction showing specific and sensitive amplification anddetection.

The specificity of the miR-802 assay for mature sequences was tested onmature miR-802 and pre-miR-802; a hairpin precursor in vitro transcriptof the mature miR. Mature processed miRNAs have different cellularfunctions from their hairpin precursors, and thus it is desirable thatthey are differentiated by a detection system. Target nucleic acid wasprepared at 5×10.sup.7 copies and 10-fold serial dilutions down to 5copies were made. Non-target nucleic acid was prepared at 5×10.sup.7copies and 10-fold serial dilutions down to 5 copies were made. Negativecontrols were water. Amplification reactions were preformed intriplicate and include one of the target nucleic acids and one of thenon-target nucleic acids each at equal amounts. As shown in FIG. 4, themiR-802 assay showed good discrimination of the mature miR-802 templatefrom its corresponding hairpin precursor, with differential detectionbeing between 50 and 500 copies of each species. The amplification anddetection oligomers and methods provide sensitive and specific resultsin the presence of unrelated non-target nucleic acids and in thepresence of precursor nucleic acid molecules.

Because miRNA family members can differ in sequence by one or morenucleotides, we next designed a system to demonstrate the sequencespecificity of our real-time TMA assay against related non-targetnucleic acids. A real-time TMA assay to specifically detect miR-34b andwas tested against miR-34 family members miR-34a, miR-34c and othernon-target miRNA let-7d. Target nucleic acids were as follows: SEQ IDNO:29, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:27, respectively.Amplification and detection oligomers were SEQ ID NOS:9, 15, 20 and 21.Target nucleic acids were prepared at 5×10.sup.7 copies and 10-foldserial dilutions down to 5 copies was made. Negative controls werewater. Amplification reactions were preformed in triplicate and includeone of the target or non-target nucleic acids. As shown in FIG. 5 andtable 6, the miR-34b assay showed template-dependent amplification atabout 20 minutes with 5×10.sup.7 input copies per reaction using miR-34bsynthetic miRNA template compared to synthetic mature miRNA familymembers miR-34a and miR-34c and the unrelated miRNA let-7d. Somecross-reactivity was seen with 5×10.sup.7 copies per reaction of therelated species miR-34c at about 42 minutes (FIG. 5B), which isapproximately the time that background signal was detected in the notemplate control reaction (about 45 minutes). Thus, the analyticalsensitivity of this assay for the miR-34b species was less than 50,000copies per reaction in the presence of related target nucleic acids. Theamplification and detection oligomers and methods provide sensitive andspecific results in the presence of related non-target nucleic acid.

TABLE 6 Average TTime Nucleic Acid (amount) (minutes) let7d (5 copies)44.6 let7d (50 copies) 41.7 let7d (500 copies) 47.2 let7d(5,000copies) >50 let7d (50,000 copies) 44.2 let7d (5 × 10.sup5) 44.4 let7d (5× 10.sup6) 45.3 let7d (5 × 10.sup7) 47.5 mir34a (5 copies) >50 mir34a(50 copies) 44.9 mir34a (500 copies) >50 mir34a(5,000 copies) 45.0mir34a (50,000 copies) 42.8 mir34a (5 × 10.sup5) 44.1 mir34a (5 ×10.sup6) 47.1 mir34a (5 × 10.sup7) >50 mir34b (5 copies) 47.7 mir34b (50copies) 44.6 mir34b (500 copies) 46.6 mir34b(5,000 copies) 42.1 mir34b(50,000 copies) 38.0 mir34b (5 × 10.sup5) 32.1 mir34b (5 × 10.sup6) 26.3mir34b (5 × 10.sup7) 19.9 mir34c (5 copies) 44.8 mir34c (50 copies) 44.9mir34c (500 copies) 49.0 mir34c(5,000 copies) 41.4 mir34c (50,000copies) >50 mir34c (5 × 10.sup5) 43.9 mir34c (5 × 10.sup6) 43.7 mir34c(5 × 10.sup7) 42.1

The need to discriminate between closely related family members ishighly desirable for a diagnostic assay. The above data show that thecurrent compositions and methods provide a highly selective assay withsensitivities from about 5 to about 5000 copies of target nucleic acidin the presence of unrelated target nucleic acids, related precursornucleic acids and related mature miRs. To improve this performance inthe presence of related nucleic acid molecules, the length of the3′-extender primer was increased. In some instances, this resulted inelevated background signals, presumably due to increased sequenceoverlap with the T7-provider oligonucleotide (i.e. resulting innon-specific amplification). Use of a target capture oligomer tospecifically hybridize and isolate target nucleic acids further improvesassay sensitivity and specificity.

A particular advantage of these assays is that FFPE sections, inaddition to fresh or frozen tissue, can be placed directly in STM forconvenient transport and/or storage. The miRNA targets can then bespecifically captured directly from the STM buffer without the need foradditional RNA purification.

Example 8 miR-21, miR-182, miR-221 and miR-22 for Diagnostic Panel

Several miRNAs were prioritized based on their relative expressionlevels determined in our microarray assay (Example 6). Top candidateswere further selected on criterion such as chromosomal location,predicted biological contexts, and published studies. In Example 6, weexamined miRNA profiles of human prostate cancer xenografts to determinewhich miRNAs could be detected in moderate to high abundance and whichwere differentially expressed compared to normal prostate tissue. Basedon this analysis, we concluded that miR-802 was not highly expressed inthe xenografts and was not differentially expressed between normal andtumor samples. In contrast, we identified several miRNAs that showedhigh levels of expression and/or were differentially expressed in thexenografts. Four of these miRNAs, miR-221, miR-222, miR-21 and miR-182,were selected for detection using real-time TMA assays. Surprisingly,miR221 and miR222 showed high expression across all xenographs, but wasstrongly downregulated in prostate tumor tissue compared to normaltissue, (see Siva et al, Mol. Cancer (2009), 8:17). Target nucleic acidswere as follows: SEQ ID NO:38 (miR-21), SEQ ID NO:41 (miR-182), SEQ IDNO:44 (miR-221) and SEQ ID NO:47 (miR-222). Amplification and detectionoligomers were as follows: for miR-21, SEQ ID NOS:8, 14, 20, 21; formiR-182, 10, 16, 20, 21; for miR-221, SEQ ID NOS:11, 17, 20, 21; and formiR-222, 12, 18, 20, 21. Target nucleic acids were prepared at5×10.sup.7 copies and 10-fold serial dilutions down to 5 copies weremade. Negative controls were water. Amplification reactions werepreformed in triplicate and include one of the target nucleic acids. Asshown in FIG. 6A, miR-221, miR-222, miR-21 and miR-182 all showed goodanalytical performance. In a second assay, selectivity for miR-221 inthe presence of miR-222 mir-30b (SEQ ID NO:50) and miR-802 was shown.Amplification and detection oligomers were SEQ ID NOS:11, 17, 20, 21.Target nucleic acid was prepared at 5 x 10.sup.7 copies and 10-foldserial dilutions down to 5 copies were made. Non-target nucleic acid wasprepared at 5×10.sup.7 copies and 10-fold serial dilutions down to 5copies were made. Negative controls were water. Amplification reactionswere preformed in triplicate and include one of the target nucleic acidsand one of the non-target nucleic acids each at equal amounts. Resultsare shown in FIG. 6B, and are analogous to our previous demonstrationfor the miR-802 assay. The assay showed good discrimination for thetarget nucleic acid, with differential detection being between 50 and500 copies of miR-221 and the non-target nucleic acid, includingmiR-222. The amplification and detection oligomers and methods providesensitive and specific results in the presence of unrelated non-targetnucleic acids and in the presence of precursor nucleic acid molecules.

TMA assays for these miRNAs miR-21, miR-182, miR-221 and miR-222 showedaccurate and reproducible quantitation using 10 ng of total RNA fromthese tissues. The TMA assay showed good specificity in the presence ofrelated target nucleic acids. Similar results were also obtained whenthese miRNAs were detected directly from STM extracted clinical FFPEspecimens and urine sediments (data not shown).

Example 9 miRNA Copy Number in Prostate Cancer Cell Lines and Xenografts

To assess the efficiency of our real-time TMA assay for capturing anddetecting miRNA from a complex mixture, known quantities of syntheticmiR-221 were added to either Solution Transport Medium (STM) or STMcontaining total RNA extracted from LNCaP or VCaP cells (10 ng total RNAper reaction), both of which were shown separately to contain low orundetectable levels of endogenous miR-221. Control reactions were runwith synthetic miR-221 transcripts added directly to amplificationreagent (without target capture and without the addition of total RNA).Capture and amplification efficiencies for known input copy numbers ofsynthetic miR-221 were similar in the presence or absence of totalcellular RNA (FIG. 7A, curves for “LNCaP/STM” and “VCaP/STM” compared to“pure STM”), indicating that the integrated target capture andamplification/detection compositions and methods provide highly accuratedetection data, even for target nucleic acids present in an abundant andmixed population of non-target nucleic acids. Moreover, the integratedtarget capture and amplification/detection performed only slightly lessefficiently than did the control reaction wherein the target nucleicacid was added directly into the amplification reaction mixture. (FIG.7A, “control amp”). Thus, the integrated target capture andamplification/detection reaction is a very robust system for detectingeven minute amounts of target nucleic acids in the presence of anabundance of non-target nucleic acids.

Using a commercial TaqMan qRT-PCR assay and the above TMA assay,endogenous expression levels of miR-221 were measured in the prostatecancer cell lines LNCaP, PC-3, DU145 and VCaP (FIG. 7B). We measuredrelatively low or undetectable levels of endogenous miR-221 in LNCaP andVCaP cells as reported above. Results from the TaqMan qRT-PCR assaysshowed 3005 copies of miR-221 per PC-3 cell and 293 copies per DU145cell, while the TMA with integrated target capture showed 2414 and 215copies. (FIGS. 7B-C) Differences in data could be due to false positiveresults in the commercial RT-PCR assay, which did not include a targetcapture step. However, additional studies should be performed to furtherunderstand the mechanism behind the high positive results for thecommercial kits. These tests were also performed on total RNA isolatedfrom prostate cancer xenograft samples (FIG. 7D). Endogenous expressionlevels of miR-21, miR-182, miR-221 and miR-222 were measured using a TMAassay with integrated target capture, amplification and detection. Ourresults for miR-21 expression in tumor xenograft samples showed about a5- to 25-fold upregulation in these tissues compared to normal sampleswith copy numbers ranging from 5000 to 35,000 copies per cell (FIG. 8A).Due to its implied role in cancer and its high level of expression inprostate tumor compared to normal prostate samples, miR-21 may havevalue in distinguishing tumor from normal tissue. These results alsoshowed miR-182 was highly upregulated in the majority of tumor tissuescompared to normal samples (FIG. 8B). We did not observe a significantdifference in miR-221 or miR-222 expression in the three matched pairsof androgen dependent and androgen independent xenograft lines (LuCaP23.1/23.1 AI, 35135V, and 96/96AI, FIG. 8B). Expression levels formiR-221 and miR-222 in patient biopsy cores, metastatic lymph nodespecimens, and non-malignant prostate tissues suggest that these miRNAscould be associated with invasive prostate cancer. Based on these andother observations, miR-221 and miR-222 appear to be promising biomarkercandidates for discriminating metastatic subtypes.

TABLE 7 Precursor, Mature and Minor miR Sequences Discussed hereinmiRBase SEQ ID NO Seq 5 → 3 MiR name Accession Number 27agagguaguagguugcauaguu let-7d MIMAT0000065 28 GUGCUCGGUUUGUAGGCAGUGUCpre-miR-34b MI0000742 AUUAGCUGAUUGUACUGUGGUGG UUACAAUCACUAACUCCACUGCCAUCAAAACAAGGCAC 29 caaucacuaacuccacugccau miR-34b MIMAT0004676 30uaggcagugucauuagcugauug minor miR-34b MIMAT0000685 31aaaaguaauugugguuuuugcc miR-548d-5p MIMAT0004812 32caaaaaccacaguuucuuuugc miR-548d-3p MIMAT0003323 33GUUCUGUUAUUUGCAGUCAGUAA pre-miR-802 MI0003906 CAAAGAUUCAUCCUUGUGUCCAUCAUGCAACAAGGAGAAUCUUUGU CACUUAGUGUAAUUAAUAGCUGG AC 34caguaacaaagauucauccuugu miR-802 MIMAT0004185 35 uggcagugucuuagcugguugumiR-34a MIMAT0000255 36 aggcaguguaguuagcugauugc miR-34c MIMAT0000686 37UGUCGGGUAGCUUAUCAGACUGA pre-miR-21 MI0000077 UGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUG ACA 38 UAGCUUAUCAGACUGAUGUUGA miR-21MIMAT0000076 39 CAACACCAGUCGAUGGGCUGU minor miR-21 MIMAT0004494 40GAGCUGCUUGCCUCCCCCCGUUU pre-miR-182 MI0000272 UUGGCAAUGGUAGAACUCACACUGGUGAGGUAACAGGAUCCGGUGG UUCUAGACUUGCCAACUAUGGGG CGAGGACUCAGCCGGCAC 41uuuggcaaugguagaacucacac miR-182 MIMAT0000259 u 42 ugguucuagacuugccaacuaminor miR-182 MIMAT0000260 43 UGAACAUCCAGGUCUGGGGCAUG pre-miR-221MI0000298 AACCUGGCAUACAAUGUAGAUUU CUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCU ACCUGGAAACAUGUUCUC 44 AGCUACAUUGUCUGCUGGGUUUCmiR-221 MIMAT0000278 45 accuggcauacaauguagauuu minor miR-221MIMAT0004568 46 GCUGCUGGAAGGUGUAGGUACCC pre-mir-222 MI0000299UCAAUGGCUCAGUAGCCAGUGUA GAUCCUGUCUUUCGUAAUCAGCA GCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU 47 agcuacaucuggcuacugggu miR-222 MIMAT0000279 48cucaguagccaguguagauccu minor miR-222 MIMAT0004569 50uguaaacauccuacacucagcu miR30b MIMAT0000420

1. A multiplex amplification method for specifically detecting thepresence of target nucleic acids in a sample comprising the steps of:(a) providing a target capture probe for capturing a miR-221 targetnucleic acid and a target capture probe for capturing a miR-182 targetnucleic acid, wherein the target capture probes are each made up of aninternal target hybridizing sequence flanked by a capture region and aterminal region, wherein the terminal region binds to the capture regionto form a double-stranded stem portion of the hairpin structure, whereinthe target hybridizing region forms a single-stranded loop portion ofthe hairpin structure, and wherein the target hybridizing sequence ofthe miR-221 target capture probe is substantially complementary to allor a portion of a miR-221 sequence and the target hybridizing sequenceof the miR-182 target capture probe is substantially complementary toall or a portion of a miR-182 sequence, (b) mixing a sample suspected ofcontaining a miR-221 target nucleic acid and a miR-182 target nucleicacid with the capture probes, (c) specifically hybridizing the targethybridizing sequence of the capture probes to a target sequence in theirrespective target nucleic acids, (d) binding the capture regions to animmobilized probe attached to a capture support by binding togethermembers of a specific binding pair, thereby forming a capture hybridmade up of the miR-221 target nucleic acid, the capture probe and theimmobilized probe attached to the capture support, and a capture hybridmade up of the miR-182 target nucleic acid, the capture probe, and theimmobilized probe attached to the capture support, (e) separating thecapture hybrids from other components in the sample, (f) releasing themiR-221 and miR-182 target nucleic acids from the capture hybrids andproviding a set of conditions wherein the target capture probes arepartially double-stranded hairpin structures, (g) providing to themixture comprising the released miR-221 and miR-182 target nucleic acidsand the target capture probes a plurality of amplification oligomers,the plurality comprising (i) at least a first amplification oligomer anda promoter-based oligomer for specifically hybridizing to a miR-221target nucleic acid or cDNA strand thereof, wherein the firstamplification oligomer comprises a target hybridizing sequence, aportion of which is substantially identical to a portion of the targethybridizing region of the miR-221 target capture probe, and wherein thepromoter-based oligomer comprises a target hybridizing sequence, aportion of which is substantially complementary to a portion of thetarget hybridizing region of the miR-221 target capture probe, and (ii)at least a first amplification oligomer and a promoter-based oligomerfor hybridizing to a miR-182 target nucleic acid or cDNA strand thereof,wherein the first amplification oligomer comprises a target hybridizingsequence, a portion of which is substantially identical to a portion ofthe target hybridizing region of the miR-182 target capture probe, andwherein the promoter-based oligomer comprises a target hybridizingsequence, a portion of which is substantially complementary to a portionof the target hybridizing region of the miR-182 target capture probe;(h) providing a set of conditions wherein the first amplificationoligomers hybridizes to their respective target nucleic acids, (i)performing an amplification reaction using the plurality ofamplification oligomers to generate amplification products from themiR-221 and miR-182 target nucleic acids; and (j) detecting theamplification products to indicate presence of miR-221 target nucleicacid, miR-182 target nucleic acid or both in the sample.
 2. The methodof claim 1, wherein binding the capture region to the immobilized proberesults from hybridizing complementary sequences of the capture regionand the immobilized probe.
 3. The method of claim 1, wherein the captureregion is a nucleotide sequence selected from the group consisting of:dT.sub.0-3A.sub.10-30.
 4. The method of claim 1, wherein the captureregion is dA.sub.12.
 5. The method of claim 1, wherein releasing themiR-221 and miR-182 target nucleic acids from the capture hybridsfurther releases the capture probes from the immobilized probes.
 6. Themethod of claim 1, wherein the target capture probe that specificallyhybridizes to a miR-221 target nucleic acid has a nucleotide sequencethat is at least 95% identical to SEQ ID NO:24.
 7. The method of claim1, wherein the target capture probe that specifically hybridizes to amiR-221 target nucleic acid has a target hybridizing sequence that is atleast 95% complementary to SEQ ID NO:44.
 8. The method of claim 7,wherein the target capture probe that specifically hybridizes to amiR-221 target nucleic acid is SEQ ID NO:24.
 9. The method of claim 1,wherein the target capture probe that specifically hybridizes to amiR-182 target nucleic acid has a nucleotide sequence that is at least95% identical to SEQ ID NO:23.
 10. The method of claim 6, wherein thetarget capture probe configured to capture a miR-182 target nucleic acidcomprises a target hybridizing sequence that is substantiallycomplementary to SEQ ID NO:41.
 11. The method of claim 6, wherein thetarget capture probe that specifically hybridizes to a miR-182 targetnucleic acid has a nucleotide sequence that is at least 95% identical toSEQ ID NO:23.
 12. The method of claim 6, wherein the target captureprobe that specifically hybridizes to a miR-182 target nucleic acid isSEQ ID NO:23.
 13. The method of claim 1, wherein the target captureprobe that specifically hybridizes to a miR-182 target nucleic acid isSEQ ID NO:23.
 14. The method of claim 1, wherein the first amplificationoligomer that specifically hybridizes to miR-221 is an extender primercomprising a 3′ target hybridizing sequence and a 5′ extension nucleicacid sequence, wherein the 3′ target hybridizing sequence issubstantially complementary to about 9 or more contiguous nucleic acidsat the 3′ end of SEQ ID NO:44.
 15. The method of claim 14, wherein themiR-221 first amplification.oligomer has an extension nucleic acidsequence that is at least 95% identical to SEQ ID NO:49.
 16. The methodof 1, wherein the first amplification oligomer that specificallyhybridizes to miR-221 is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially identical to about 9or more contiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-221 target capture probe.
 17. The method of claim16, wherein the miR-221 target capture probe is SEQ ID NO:24.
 18. Themethod of claim 16, wherein the miR-221 first amplification oligomer hasan extension nucleic acid sequence that is at least 95% identical to SEQID NO:49.
 19. The method of claim 1, wherein the first amplificationoligomer is SEQ ID NO:17.
 20. The method of claim 1, wherein the firstamplification oligomer that specifically hybridizes to miR-182 is anextender primer comprising a 3′ target hybridizing sequence and a 5′extension nucleic acid sequence, wherein the 3′ target hybridizingsequence is substantially complementary to about 9 or more contiguousnucleic acids at the 3′ end of SEQ ID NO:41.
 21. The method of claim 20,wherein the miR-221 first amplification oligomer has an extensionnucleic acid sequence that is at least 95% identical to SEQ ID NO:49.22. The method of 1, wherein the first amplification oligomer thatspecifically hybridizes to miR-182 is an extender primer comprising a 3′target hybridizing sequence and a 5′ extension nucleic acid sequence,wherein the 3′ target hybridizing sequence is substantially identical toabout 9 or more contiguous nucleic acids at the 5′ end of the targethybridizing sequence of the miR-182 target capture probe.
 23. The methodof claim 22, wherein the miR-182 target capture probe is SEQ ID NO:23.24. The method of claim 22, wherein the miR-182 first amplificationoligomer has an extension nucleic acid sequence that is at least 95%identical to SEQ ID NO:49.
 25. The method of claim 1, wherein the firstamplification oligomer is SEQ ID NO:16.
 26. The method of claim 19,wherein the first amplification oligomer that specifically hybridizes tomiR-182 is an extender primer comprising a 3′ target hybridizingsequence and a 5′ extension nucleic acid sequence, wherein the 3′ targethybridizing sequence is substantially complementary to about 9 or morecontiguous nucleic acids at the 3′ end of SEQ ID NO:41.
 27. The methodof claim 1, wherein the promoter based oligomer that specificallyhybridizes to a complement of the miR-221 target nucleic acid comprisesa target hybridizing sequence that is substantially complementary to 9or more contiguous nucleic acids on 3′ end of the miR-221 target captureprobe target hybridizing region.
 28. The method of claim 1, wherein thepromoter based amplification oligomer that specifically hybridizes to acomplement of the miR-221 target nucleic acid is SEQ ID NO:11.
 29. Themethod of claim 28, wherein the promoter based oligomer thatspecifically hybridizes to a complement of the miR-221 target nucleicacid comprises a target hybridizing sequence that is substantiallycomplementary to 9 or more contiguous nucleic acids on 3′ end of themiR-221 target capture probe target hybridizing region.
 30. The methodof claim 1, wherein the promoter based oligomer that specificallyhybridizes to a complement of the miR-182 target nucleic acid comprisesa target hybridizing sequence that is substantially complementary to 9or more contiguous nucleic acids on 3′ end of the miR-182 target captureprobe target hybridizing region.
 31. The method of claim 1, wherein thepromoter based amplification oligomer that specifically hybridizes to acomplement of the miR-182 target nucleic acid is SEQ ID NO:10.
 32. Themethod of claim 31, wherein the promoter based oligomer thatspecifically hybridizes to a complement of the miR-221 target nucleicacid comprises a target hybridizing sequence that is substantiallycomplementary to 9 or more contiguous nucleic acids on 3′ end of themiR-221 target capture probe target hybridizing region.
 33. The methodof claim 1, further comprising a reverse primer oligomer.
 34. The methodof claim 1, wherein the first amplification oligomer that specificallyhybridizes to miR-221 is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, andwherein at or after step (g) a reverse primer is provided.
 35. Themethod of claim 34, wherein the reverse primer is configured tohybridize to a portion of the complement of the extension nucleic acid.36. The method of claim 1, wherein the first amplification oligomer thatspecifically hybridizes to miR-182 is an extender primer comprising a 3′target hybridizing sequence and a 5′ extension nucleic acid sequence,and wherein at or after step (g) a reverse primer is provided.
 37. Themethod of claim 36, wherein the reverse primer is configured tohybridize to a portion of the complement of the extension nucleic acid.38. The method of claim 34, wherein the first amplification oligomerthat specifically hybridizes to miR-182 is an extender primer comprisinga 3′ target hybridizing sequence and a 5′ extension nucleic acidsequence, and wherein at or after step (g) a reverse primer is provided.39. The method of claim 38, wherein the 5′ extension nucleic acidsequences for both the miR-221 extender primer and the miR-182 extenderprimer are substantially identical and the reverse primer is a universalprimer configured to hybridize to a portion of the complement of theextension nucleic acid.
 40. The method of claim 38, wherein the 5′extension nucleic acid sequences for both the miR-221 extender primerand the miR-182 extender primer comprise a complement of a reverseprimer target sequence.
 41. The method of claim 40, wherein thecomplement reverse primer target sequences on the miR-221 extenderprimer and the miR-182 extender primer are substantially identical, andwherein the reverse primer is a universal primer.
 42. The method ofclaim 33, wherein the reverse primer is SEQ ID NO:20.
 43. The method ofclaim 39, wherein the reverse primer is SEQ ID NO:20.
 42. The method ofclaim 41, wherein the reverse primer is SEQ ID NO:20.
 43. The method ofclaim 1, wherein the amplification reaction is performed in the presenceof target capture probe and wherein the amplification reactionconditions are configured to maintain the target capture probe in ahairpin formation.
 44. The method of claim 1, wherein the amplificationreaction is an isothermal amplification reaction.
 45. The method ofclaim 1, wherein amplification products are detected using a detectionprobe oligomer.
 46. The method of claim 45, wherein the detection probeoligomer is a linear detection probe.
 47. The method of claim 45,wherein the detection probe oligomer is a hairpin detection probe. 48.The method of 45, wherein the detection probe oligomer is labeled. 49.The method of claim 45, wherein the detection probe oligomer is duallabeled.
 50. The method of claim 1, wherein the detection of the targetnucleic acid is a real-time detection.
 51. The method claim 1, whereinthe detection of the target nucleic acid is useful for diagnosingcancer, providing a prognosis relating to a cancer, monitoring theeffectiveness of a cancer treatment, or a combination thereof.
 52. Themethod of claim 51, wherein the cancer is prostrate cancer.
 53. A methodof detecting the presence of a target nucleic acid in a samplecomprising the steps of: (a) providing a target capture probe that formsunder hybridizing conditions a partially double-stranded hairpinstructure made up of an internal target hybridizing sequence flanked bya capture region and a terminal region, wherein the terminal regionbinds to the capture region to form a double-stranded stem portion ofthe hairpin structure, and wherein the target hybridizing region forms asingle-stranded loop portion of the hairpin structure, (b) mixing asample suspected of containing a miR-221 target nucleic acid with thecapture probe, (c) specifically hybridizing the target hybridizingsequence of the capture probe to a target sequence in the miR-221 targetnucleic acid, (d) binding the capture region to an immobilized probeattached to a capture support by binding together members of a specificbinding pair, thereby forming a capture hybrid made up of the miR-221target nucleic acid, the capture probe, and the immobilized probeattached to the capture support, (e) separating the capture hybridattached to the capture support from other components in the sample, (f)releasing the miR-221 target nucleic acid from the capture hybrid, (g)providing to the mixture comprising the released miR-221 target nucleicacid and the target capture probe at least a first amplificationoligomer comprising a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe target capture probe; (h) providing a set of conditions wherein thefirst amplification oligomer hybridizes to the miR-221 target nucleicacid and an amplification reaction takes place, and wherein the targetcapture probe is in a partially double stranded hairpin formation, (i)generating a detectable amplification product in an amplificationreaction wherein the first amplification oligomer generates a cDNAstrand from the miR-221 target nucleic acid; and (j) detecting theamplification product to indicate presence of miR-221 target nucleicacid in the sample.
 54. A method of detecting the presence of a targetnucleic acid in a sample comprising the steps of: (a) providing a targetcapture probe that forms under hybridizing conditions a partiallydouble-stranded hairpin structure made up of an internal targethybridizing sequence flanked by a capture region and a terminal region,wherein the terminal region binds to the capture region to form adouble-stranded stem portion of the hairpin structure, and wherein thetarget hybridizing region forms a single-stranded loop portion of thehairpin structure, (b) mixing a sample suspected of containing a miR-221target nucleic acid with the capture probe, (c) specifically hybridizingthe target hybridizing sequence of the capture probe to a targetsequence in the miR-221 target nucleic acid, (d) binding the captureregion to an immobilized probe attached to a capture support by bindingtogether members of a specific binding pair, thereby forming a capturehybrid made up of the miR-221 target nucleic acid, the capture probe,and the immobilized probe attached to the capture support, (e)separating the capture hybrid attached to the capture support from othercomponents in the sample, (f) releasing the miR-221 target nucleic acidfrom the capture hybrid and providing a set of conditions wherein thetarget capture probe is a partially double-stranded hairpin structure,(g) providing to the mixture comprising the released miR-221 targetnucleic acid and the target capture probe a plurality of amplificationoligomers, the plurality comprising at least a first amplificationoligomer and a promoter-based oligomer, wherein the first amplificationoligomer comprises a target hybridizing sequence, a portion of which issubstantially identical to a portion of the target hybridizing region ofthe target capture probe, and wherein the promoter-based oligomercomprises a target hybridizing sequence, a portion of which issubstantially complementary to a portion of the target hybridizingregion of the target capture probe; (h) providing a set of conditionswherein the first amplification oligomer hybridizes to the miR-221target nucleic acid in the presence of the hairpin capture probe, (i)performing an amplification reaction using the plurality ofamplification oligomers to generate amplification product from themiR-221 target nucleic acid; and (j) detecting the amplification productto indicate presence of miR-221 target nucleic acid in the sample. 55.An amplification reaction mixture comprising: (a) a miR-221 targetcapture probe that forms under hybridizing conditions at least apartially double-stranded structure that is a hairpin structure made upof a contiguous linear sequence that includes an internal targethybridizing sequence that is substantially complementary to all or aportion of SEQ ID NO:44 and that forms a single-stranded loop portion ofthe hairpin structure, wherein the internal target hybridizing sequenceis flanked by a capture region and by a terminal region that binds tothe capture region to form a double-stranded stem portion of the hairpinstructure and wherein the capture region further comprises asubstantially homopolymeric nucleic acid sequence and the terminalregion further comprises a substantially homopolymeric nucleic acidsequence that is complementary to the capture region sequence; and (b) afirst amplification oligomer configured to hybridize to a miR-221 targetnucleic acid that is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially identical to about 9or more contiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-221 target capture probe.
 56. The amplificationreaction mixture of claim 55, further comprising a miR-182 targetcapture probe that forms under hybridizing conditions at least apartially double-stranded structure that is a hairpin structure made upof a contiguous linear sequence that includes an internal targethybridizing sequence that is substantially complementary to all or aportion of SEQ ID NO:41 and that forms a single-stranded loop portion ofthe hairpin structure, wherein the internal target hybridizing sequenceis flanked by a capture region and by a terminal region that binds tothe capture region to form a double-stranded stem portion of the hairpinstructure and wherein the capture region further comprises asubstantially homopolymeric nucleic acid sequence and the terminalregion further comprises a substantially homopolymeric nucleic acidsequence that is complementary to the capture region sequence.
 57. Theamplification reaction mixture of claim 55, further comprising a firstamplification oligomer configured to hybridize to a miR-182 targetnucleic acid that is an extender primer comprising a 3′ targethybridizing sequence and a 5′ extension nucleic acid sequence, whereinthe 3′ target hybridizing sequence is substantially identical to about 9or more contiguous nucleic acids at the 5′ end of the target hybridizingsequence of the miR-182 target capture probe.
 58. The amplificationreaction mixture of claim 55, further comprising a promoter basedoligomer configured to specifically hybridize to a miR-221 targetnucleic acid, that comprises a target hybridizing sequence that issubstantially complementary to 12 or more contiguous nucleic acids on 3′end of the miR-221 target capture probe.
 59. The amplification reactionmixture of claim 58, wherein the miR-221 promoter based oligomer is atleast 95% identical to SEQ ID NO:11.
 60. The amplification reactionmixture of claim 58, wherein the miR-221 promoter based oligomer is SEQID NO:11.
 61. The amplification reaction mixture of claim 55, whereinthe miR-221 target capture probe is at least 95% identical to SEQ IDNO:24.
 62. The amplification reaction mixture of claim 55, wherein themiR-221 target capture probe is SEQ ID NO:24.
 63. The amplificationreaction mixture of claim 55, wherein the miR-221 extender primer is atleast 95% identical to SEQ ID NO:17.
 64. The amplification reactionmixture of any one of claims 51 to 56, wherein the miR-221 extenderprimer is SEQ ID NO:17.
 65. The amplification reaction mixture of claim56, further comprising a promoter based oligomer configured tospecifically hybridize to a miR-182 target nucleic acid, that comprisesa target hybridizing sequence that is substantially complementary to 12or more contiguous nucleic acids on 3′ end of the miR-182 target captureprobe.
 66. The amplification reaction mixture of claim 65, wherein thepromoter based oligomer is at least 95% identical to SEQ ID NO:10. 67.The amplification reaction mixture of claim 65, wherein the promoterbased oligomer is SEQ ID NO:10.
 68. The amplification reaction mixtureof claim 56, wherein the miR-182 target capture probe is at least 95%identical to SEQ ID NO:23.
 69. The amplification reaction mixture ofclaim 56, wherein the miR-182 target capture probe is SEQ ID NO:23. 70.The amplification reaction mixture of claim 57, wherein the miR-182extender primer is at least 95% identical to SEQ ID NO:16.
 71. Theamplification reaction mixture of any one of claims 70, wherein themiR-182 extender primer is SEQ NO:16.
 72. The amplification reactionmixture of claim 55, further comprising a reverse primer.
 73. Theamplification reaction mixture of claim 56, further comprising auniversal reverse primer.
 74. The amplification reaction mixture ofclaim 55, wherein the target capture probe comprises at least one 2′-OMeresidue in the target hybridizing sequence.
 75. The amplificationreaction mixture of claim 56, wherein the target capture probe comprisesat least one 2′-OMe residue in the target hybridizing sequence.